Evidence of the Physical Interaction between Rpl22 and the Transposable Element Doc5, a Heterochromatic Transposon of Drosophila melanogaster

Abstract

Chromatin is a highly dynamic biological entity that allows for both the control of gene expression and the stabilization of chromosomal domains. Given the high degree of plasticity observed in model and non-model organisms, it is not surprising that new chromatin components are frequently described. In this work, we tested the hypothesis that the remnants of the Doc5 transposable element, which retains a heterochromatin insertion pattern in the melanogaster species complex, can be bound by chromatin proteins, and thus be involved in the organization of heterochromatic domains. Using the Yeast One Hybrid approach, we found Rpl22 as a potential interacting protein of Doc5. We further tested in vitro the observed interaction through Electrophoretic Mobility Shift Assay, uncovering that the N-terminal portion of the protein is sufficient to interact with Doc5. However, in situ localization of the native protein failed to detect Rpl22 association with chromatin. The results obtained are discussed in the light of the current knowledge on the extra-ribosomal role of ribosomal protein in eukaryotes, which suggests a possible role of Rpl22 in the determination of the heterochromatin in Drosophila.

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Article
Evidence of the Physical Interaction between Rpl22
and the Transposable Element Doc5, a Heterochromatic
Transposon of Drosophila melanogaster
Maria Francesca Berloco 1, †, Crescenzio Francesco Minervini 2 ,† , Roberta Moschetti 1, Antonio Palazzo 1,
Luigi Viggiano 1,*, ‡ and RenéMassimiliano Marsano 1, *,‡ ,§


Citation: Berloco, M.F.; Minervini,
C.F.; Moschetti, R.; Palazzo, A.;
Viggiano, L.; Marsano, R.M. Evidence
of the Physical Interaction between
Rpl22 and the Transposable Element
Doc5, a Heterochromatic Transposon
of Drosophila melanogaster.Genes 2021,
12, 1997. https://doi.org/
10.3390/genes12121997
Academic Editor: Miroslav Plohl
Received: 6 November 2021
Accepted: 12 December 2021
Published: 16 December 2021
Publisher’s Note: MDPI stays neutral
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Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1Department of Biology, University of Bari “Aldo Moro”, 70126 Bari, Italy;
mariafrancesca.berloco@uniba.it (M.F.B.); roberta.moschetti@uniba.it (R.M.); antonio.palazzo@uniba.it (A.P.)
2
Department of Emergency and Organ Transplantation (D.E.T.O.), Hematology and Stem Cell Transplantation
Unit, University of Bari “Aldo Moro”, 70124 Bari, Italy; crescenziofrancesco.minervini@uniba.it
*Correspondence: luigi.viggiano@uniba.it (L.V.); renemassimiliano.marsano@uniba.it (R.M.M.)
† joint first authors.
‡ joint corresponding authors.
§ Former affiliation: Department of Genetics Anthropology Evolution, University of Parma, Parco Area delle
Scienze 11/A, 43124 Parma, Italy.
Abstract:
Chromatin is a highly dynamic biological entity that allows for both the control of gene
expression and the stabilization of chromosomal domains. Given the high degree of plasticity ob-
served in model and non-model organisms, it is not surprising that new chromatin components are
frequently described. In this work, we tested the hypothesis that the remnants of the Doc5 transpos-
able element, which retains a heterochromatin insertion pattern in the melanogaster species complex,
can be bound by chromatin proteins, and thus be involved in the organization of heterochromatic
domains. Using the Yeast One Hybrid approach, we found Rpl22 as a potential interacting protein of
Doc5. We further tested
in vitro
the observed interaction through Electrophoretic Mobility Shift Assay,
uncovering that the N-terminal portion of the protein is sufficient to interact with Doc5. However,
in situ localization of the native protein failed to detect Rpl22 association with chromatin. The
results obtained are discussed in the light of the current knowledge on the extra-ribosomal role of
ribosomal protein in eukaryotes, which suggests a possible role of Rpl22 in the determination of the
heterochromatin in Drosophila.
Keywords:
ribosomal protein; Rpl22; Drosophila; DNA–protein interaction; transposable elements;
heterochromatin; Doc5/Porto1
1. Introduction
Chromatin [
1
] is a nucleoprotein complex that plays a key role in controlling cell
behavior and chromosomal structure [
2
,
3
]. Its regulation is important in the control of
cellular events, including genome packaging, replication, recombination, DNA repair, and
transcription. The nucleosome, which comprises the four core histones (H2A, H2B, H3,
H4), wrapped around with 168 bp of DNA, and the linker histones H1 or H5 form the
chromatosome, the structural unit of the chromatin [4].
Chromatin is found in two fundamental states during the cell cycle, the loosely
condensed euchromatin and the highly compacted heterochromatin. A huge number of
DNA–protein and protein–protein interactions contribute to the maintenance of these two
structures, the plasticity of which is tightly regulated at the epigenetic level.
Many proteins act as structural components or regulators of the chromatin state,
and post-translational modifications of many chromatin components play a fundamental
role in maintaining the dynamic state of different chromatin domains. The ongoing EN-
Genes 2021,12, 1997. https://doi.org/10.3390/genes12121997 https://www.mdpi.com/journal/genes

Genes 2021,12, 1997 2 of 17
CODE projects [
5
,
6
] aim to determine the nature of the epigenetic code and to what extent
chromatin remodeling could influence the phenotypes.
Several pieces of observation suggest that ribosomal proteins (RPs) could have an
active role in chromatin dynamics. First, RNA-mediated processes have a functional role in
regulating chromatin structure and gene expression through the action of non-coding RNA
molecules [
7
–
9
]. Second, a large fraction of the expressed lncRNA interacts with ribosomes
in humans and mice (roughly 39% and 48%, respectively) [10].
Third, the presence of RPs in the nucleus is well-recognized since RPs are imported
into the nucleus and assembled into pre-ribosomes in the nucleolus [11].
Therefore, a subset of RPs could be co-opted as chromatin components to perform
additional functions under either physiological or exceptional conditions.
Heterochromatin is a partition of the eukaryotic genome, often regarded as useless
and functionless. This concept is due to its low gene density and the consequent low impact
of mutational load in this compartment on viability and fertility. The massive presence of
satellite DNA and transposons in the constitutive heterochromatin has further reinforced
this idea. However, since heterochromatin is associated with important functions and
structures of the eukaryotic chromosomes, its role has been recently re-evaluated, both
in model and non-model organisms. In D. melanogaster, several hundreds of genes have
been mapped in the constitutive heterochromatin, thus demonstrating its importance in
the physiology of cells, tissues, and organs in the fly [
12
], an observation largely supported
by classic and modern genetics studies.
Several additional features make heterochromatin a fascinating genomic compartment.
These include the massive presence of repeats and transposable elements, whose structural
and functional roles remain elusive, despite decades of studies. In this respect, noteworthy
examples still come from D. melanogaster. Several repeated loci have been characterized
so far in the heterochromatin of D. melanogaster, and some of them play an extremely
important role in determining critical phenotypes [
13
–
15
]. One of these relevant loci lies
in the h39 region, a Hoechst-bright chromosomal band adjacent to the centromere of the
second chromosome. Two well-studied satellite DNA sequences are clustered in the h39
region, the Responder locus (Rsp), and the Bari1 repeat. The Rsp locus, in combination
with the Sd euchromatic locus, constitutes the key components of one of the best-known
segregation distortion systems [
13
]. The Bari1 cluster is an array of roughly 80 copies of
the Bari1 transposon [
16
–
18
], depending on the fly strain [
18
,
19
] of the Bari1 transposon.
Elements of the Bari1 family are Tc1-like transposons that have colonized the genome of
several Drosophila species [17] and are active in the respective genomes [20,21].
The characterization of the Bari1 copy number variation in several D. melanogaster pop-
ulations [
16
,
18
,
19
] revealed that this is an extremely static array if compared to the closely
linked Rsp locus [
22
,
23
]. Considering that the Bari1 cluster origin probably dates back to
the split of the melanogaster and simulans species, approximately 5 Mya, and that it is only
present in D. melanogaster [
16
,
17
,
24
], it has been speculated that either it could be function-
ally connected to the Rsp locus or to other structural features of the h39 region. However,
the presence of a small Bari1 cluster on the X chromosome
[17,25]
and an additional small
Rsp repeat on the third chromosome [
26
] and the highly repetitive nature of the h39 region
complicate the molecular and genetic investigations of this chromosomal region.
Many transposon relics map at both sides of the Bari1 cluster in the h39 region of the
mitotic chromosomes of D. melanogaster [
27
]. A direct duplication of a 596 bp sequence
identified upstream and downstream of the Bari1 cluster is of particular interest. We hy-
pothesized that this short duplication could be the signature of the transposition-mediated
origin of the Bari1 cluster (as also hypothesized for the minor X-linked Bari1 cluster [
25
]).
Alternatively, it could have a functional role in the h39 locus or in the heterochromatin [
27
].
Since the first hypothesis seems unreliable (due to the size incompatibility and outcome of
the transposition event), in this work we tested the hypothesis that the above-described
596 bp sequence could have acquired a new function in the heterochromatin through the
binding of a chromatin protein. Here, we present evidence that the ribosomal protein Rpl22

Genes 2021,12, 1997 3 of 17
binds DNA
in vitro
, which suggests the possibility that it could be recruited as chromatin
protein. The CG7434 gene, which encodes RpL22 protein, maps on the X chromosome.
Three additional genes are present within the coding region of RpL22, two encoding snoR-
NAs (CR34590 and CR33918) and one encoding a ncoRNA (CR42491). This structure
complicates the genetic analysis of the locus, and, in fact, no genetic studies have been per-
formed focusing on this gene. At least two post-translational modification events have been
characterized, involving phosphorylation of the Ser 289 and Ser290 residues of the RpL22
in Drosophila [
28
]. Among RPs, some members of the RpL22e family have unique structural
features and several, apparently unrelated, possible functions. The Drosophila Rpl22 has
additional Ala-, Lys- and Pro-rich sequences at the amino terminus, which resembles the
carboxyl-terminal portion of histone H1 and histone H5 that have been demonstrated to
be important in genome stability [
29
]. For this reason, it has been already hypothesized
that Drosophila L22 might have two functions, namely, the role of DNA-binding similar
to histone H1 and the role of organizing the ribosome [
30
]. Moreover, as hypothesized in
previous works, any potential biological difference between Rpl22 and Rpl22-like proteins
should be ascribed to the presence of the extra N-terminal domain of Rpl22, which can be
the target of post-translational modifications [31].
We also have evidence that Rpl22 enters into the nucleus of different cell types, in
addition to what was demonstrated previously in the male germline cells [
32
]. The possible
implications in the stability of a specific heterochromatin region are discussed.
2. Materials and Methods
2.1. Plasmids Construction
The Doc5 fragment flanking the Bari1 cluster was PCR-amplified from the purified
DNA of the BACR16M08 clone (described in [
25
]) using specific primers containing EcoRI
adapters at the 5
0
end. The PCR fragment was cloned into the EcoRI site of the pGEM-T
vector (Promega) and verified by Sanger sequencing.
2.2. PCR Amplification
Primers used for PCR amplification are reported in Table 1.
Table 1. List of primers used in this study.
Primer Sequence Usage
ADread 50-CTATTCGATGATGAAGAT-30sequencing
pACT2seq 50-TACCACTACAATGGATG-30sequencing
pACT2 up 50-CTATTCGATGATGAAGATACCCCACCAAACCC-30Amplification/cloning
pACT2 low 50-GTGAACTTGCGGGGTTTTTCAGTATCTACGAT-30Amplification/cloning
His1_up 50-GAGGCCCTTTCGTCTTCAA-30Amplification/cloning
His1_low 50-CTAGGGCTTTCTGCTCTGTCATCT-30Amplification/cloning
Doc5_up 50-ACGGCTATTATTGTTTCTTATTGCT-30Amplification/cloning
Doc5_low 50-TTATCCTCATCCCTTATCCTATGT-30Amplification/cloning
pETup 50-CACCATGGCTTACCCATA-30Amplification/cloning
pETlow 50-ATAAAAGAAGGCAAAACGATG-30Amplification/cloning
H5low 50-CTAACGCAGCACGTTCTTCTT-30Amplification/cloning
L22up 50-CACCAAGGTGGTCAAGAAGAA-30Amplification/cloning
2.3. One Hybrid Screening
The one hybrid screening was performed using the Matchmaker One-Hybrid System
(Clontech, Kyoto, Japan) following the manufacturer recommendations.
ADrosophila embryonic cDNA library (cDNA pool from 0–21 h embryos of the Canton-
s strain) in the pACT2 vector (Clontech) was used for the yeast one-hybrid screens.

Genes 2021,12, 1997 4 of 17
The Doc5 sequence was subcloned into the pHISi-1 vector at the EcoRI site and into
the pLacZi vector. Both plasmids were linearized using either BamHI (pHISi-1) or NcoI
(pLacZi) and transformed in the YM4271 S. cerevisiae strain using the TRAFO system [
33
].
Recombinant colonies, carrying the integrated constructs, were selected onto selective SD
medium lacking either histidine (pHISi-1 vector) or uracil (pLacZi vector).
The background expression of the LacZ reporter was determined by a standard
β
-
galactosidase assay. Colonies were transferred to Whatman filter paper discs and lysed
with liquid nitrogen. Filters were then exposed to Z-buffer (Na
2
HPO
4·
7H
2
O 60 mM,
NaH
2
PO
4·
H
2
O 40 mM, KCl 10 mM, MgSO4 1 mM,
β
–mercaptoethanol 50 mM, pH 7)
containing X-gal (5-bromo-4-chloro-indolyl-b-D-galactopyranoside 0.33 mg/mL). Only
clones without LacZ basal expression in 8 h were selected for further analyses. These clones
were further transformed to integrate the linearized pHisi-1 vector. Background expression
of the His cassette was found to be inhibited by 15 mM 3-AT.
2.4. Protein Expression and Purification
The plasmid sets used to express proteins in E. coli (pET/RpL22 for the full-length
protein expression; pET/H5 for the H1-H5 domain expression; pET/L22 for the riboso-
mal domain expression) were constructed by PCR amplification of either the full-length,
the 5
0
-terminal or the 3
0
-terminal part of the cDNA and subsequent cloning into the
pET-200 vector.
Plasmids were transformed in chemically competent E. coli (BL21-DE3), and the
cultures were induced with 1 mM IPTG at a cell density equivalent to 0.5 OD
600
and
maintained for 2.5 h at 37
◦
C. Cells were sonicated in 25 mM HEPES (pH 7.5), 1 M NaCl,
15% glycerol, 0.25% Tween 20, 2 mM
β
-mercaptoethanol, and 1 mM PMSF. A total of 10 mM
imidazole (pH 8.0) was added to the soluble fraction before it was mixed with Ni-NTA
resin (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. The
resin was washed with sonication buffer containing 30% glycerol and 50 mM imidazole.
Bounded proteins were eluted with sonication buffer containing 300 mM imidazole and
dialyzed overnight against sonication buffer without imidazole. Purified proteins were
analyzed on 12% SDS-polyacrylamide gel. Protein concentration was determined using
the Protein Assay ESL Kit (Roche Basel, Switzerland).
2.5. Electrophoretic Mobility Shift Assay (EMSA)
In total, 5
µ
g of the pT/Doc5 plasmid was EcoRI-digested and the released fragment
was gel-purified using the QIAquick Gel Extraction Kit (Qiagen). A filling-in reaction
was performed to end-label the target DNA. A total of 50 ng of the eluted fragment was
incubated with [
α32
P]ATP (Perkin Elmer, Waltham, MA, USA), 1X Klenow reaction buffer
and 2U of Klenow fragment (Roche, Basel, Switzerland).
Labeled fragments were purified using Sephadex G50 exclusion chromatography columns.
A total of 2 ng of the labeled fragment was incubated with the appropriate protein
(either the full-length Rpl22, the H1-H5 domain or the ribosomal domain) in binding
buffer as described in [
34
] (25 mM HEPES, pH 7.6, 50 mM NaCl, 1 mM EDTA, 1 mM
DTT, 0.1 mg/mL BSA, 2.5 mM spermidine, 10% glycerol, and 0.1 mg/mL poly (dI-dC).
Competition experiments were performed using either linear pUC19 (SmaI linearized) or
sonicated
λ
phage DNA (200–1000 bp size range enrichment). The binding reaction was
started by adding the protein extract and incubated for 20 min at 25
◦
C, then loaded directly
onto 5% polyacrylamide (75:1 acrylamide:bisacrylamide) pre-run gel in 40 mM Tris–acetate,
2.5 mM EDTA (pH 7.8). Gels were run for 4.5 h at 4
◦
C at 10 V/cm and dehydrated using a
gel-dryer. DNA–protein complexes were visualized by autoradiography using a STORM
phosphorimager (Molecular Dynamics).
2.6. Fluorescence In Situ Hybridization and Immunofluorescence on Polytene Chromosomes
Fluorescence in situ hybridization experiments on polytene chromosomes were per-
formed as described in [
35
]. Polytene chromosomes were prepared from third instar larvae

Genes 2021,12, 1997 5 of 17
of D. simulans and D. sechellia, reared on standard cornmeal medium at 18
◦
C. Salivary
glands were dissected in PBS using a pair of dissection needles, fixed in 40% acetic acid, and
squashed onto microscopy slides. Probes were labeled using the nick translation method
with Cy3-dUTP, hybridized overnight at 37 ◦C.
Digital images were obtained using an Olympus epifluorescence microscope equipped
with a cooled CCD camera. Gray scale images, recording Cy3 and DAPI fluorescence, were
obtained separately using specific filters and were pseudo colored and merged to obtain
the final image using the Adobe Photoshop software.
Immunodetection experiments of Rpl22 and fibrillarin on polytene chromosomes of
the Oregon-R (wild type) were performed according to James et al. [
36
] using the polyclonal
primary anti-Rpl22 antibody (diluted 1:50) raised in rabbit (Invitrogen Carlsbad, CA, USA,
Minervini et al. submitted) and the monoclonal (G-8sc-374022 Santa Cruz Biotechnology
Inc., Dallas, TX, USA) anti-fibrillarin antibody raised in mouse. An FITC (fluorescein
isothiocyanate)-conjugated anti-rabbit Ig (whole antibody) raised in sheep (diluted 1:20)
and the Alexa Fluor 488 goat anti-mouse antibody (Life Technologies, Carlsbad, CA, USA,
1:200 dilution) were used as secondary antibodies. Following incubation, the slides were
washed three times in PBS, stained with DAPI (4,6-diamidino-2-phenilindole) at 0.01
µ
g/mL
and mounted in anti-fading medium. Immunodetection on S2R+ cells were performed as
previously described in [20,21] using the above-described antibodies.
2.7. Other Methods
Sequencing of the cloned fragments was performed at the BMR Genomics sequencing
facility (Padova, Italy).
Global alignments were performed using DNA Strider [
37
]. Local alignments were
performed using BLAST at the NCBI website.
NLS signals were searched with cNLS Mapper (http://nls-mapper.iab.keio.ac.jp/cgi-
bin/NLS_Mapper_form.cgi (accessed on 1 March 2021)) [
38
] using a cutoff score = 7 in the
entire protein sequence, and with Nucpred (https://nucpred.bioinfo.se/cgi-bin/single.cgi
(accessed on 2 March 2021)) [39].
3. Results
We have previously identified a 596 bp DNA sequence duplication (formerly named
DRM8) at both sides of the Bari1 cluster in the heterochromatin of 2R chromosome of
D. melanogaster
[
27
]. Specifically, this repetitive sequence maps in the h39 region, and it has
been proven lately to be a remnant of the Doc5/Porto1 element, a highly repeated non-LTR
retrotransposon in the heterochromatin of D. melanogaster [
40
]. The similarity between the
DRM8 sequence and the reference Doc5/Porto1 element is shown in Figure 1. Hereafter, we
will refer to this sequence as Doc5.
Several copies of the Doc5 can be found in the reference genome of D. melanogaster
(see Table 2). In silico analyses reveal that Doc5 maps exclusively in the constitutive
heterochromatin of the two major autosomes of D. melanogaster, including the centromere,
as well as at the eu-heterochromatin transition.
The heterochromatic localization of the Doc5 element is also a conserved feature in
closely related species of the melanogaster complex, such as D. simulans and D. sechellia, as
demonstrated by the results of FISH experiments on polytene chromosomes (Figure 2).

Genes 2021,12, 1997 6 of 17
Genes 2021, 12, x FOR PEER REVIEW 6 of 16
Figure 1. Comparison of the Doc5 reference sequence and the 596 bp sequence identified at both
sides of the Bari1 cluster in the h39 region of the chromosome 2 of D. melanogaster. The global se-
quence alignment (top) and the Dot-plot comparison (bottom) are shown.
Several copies of the Doc5 can be found in the reference genome of D. melanogaster
(see Table 2). In silico analyses reveal that Doc5 maps exclusively in the constitutive het-
erochromatin of the two major autosomes of D. melanogaster, including the centromere, as
well as at the eu-heterochromatin transition.
Table 2. The distribution of the Doc5 transposon in the D. melanogaster genome. The Doc5 sequence (596 bp) was used as
a query in BlastN analyses against the D. melanogaster reference genome (Release 6). The approximate map positions in
the rightmost column were inferred by comparison with the data in [12]. Only alignments longer than 100 bases are shown.
Deep het: deep heterochromatin. UNK: unknown map position.
Subject
Accession Start End Chromosome
%
Identity
Alignment
Length Evalue Bit Score
Chromosome
Map Position
NT_033779.5 23,430,152 23,429,509 2L 85.891 645 0 643 h35–36
NT_033779.5 23,037,532 23,037,717 2L 91.237 194 2.03 × 10−67 257 h35–36
NW_001845128.1 3990 4435 2CEN 100 446 0 824 deep het
NW_001845128.1 3875 3990 2CEN 100 116 1.24 × 10−54 215 deep het
NW_001844967.1 11,017 10,572 2CEN 100 446 0 824 deep het
NW_001844967.1 11,132 11,017 2CEN 100 116 1.24 × 10−54 215 deep het
NW_007931075.1 7023 6491 2CEN 82.655 565 2.43 × 10−121 436 deep het
NW_007931075.1 9914 9382 2CEN 82.655 565 2.43 × 10−121 436 deep het
NT_033778.4 396,636 397,234 2R 99.332 599 0 1083 h41–h44
NT_033778.4 165,422 164,833 2R 95.326 599 0 942 h41–h44
NT_033778.4 74,075 74,637 2R 92.833 586 0 824 h41–h44
NT_033778.4 298,134 297,689 2R 100 446 0 824 h41–h44
NT_033778.4 872,342 871,810 2R 82.655 565 2.43 × 10−121 436 h41–h44
NT_033778.4 875,233 874,701 2R 82.655 565 2.43 × 10−121 436 h41–h44
NT_033778.4 1,413,093 1,413,384 2R 91.333 300 2.47 × 10−111 403 h45
NT_033778.4 3,518,328 3,518,018 2R 88.179 313 1.95 × 10−97 357 h41–h44
NT_033778.4 5,012,337 5,012,064 2R 82.818 291 4.43 × 10−59 230 h46
Figure 1.
Comparison of the Doc5 reference sequence and the 596 bp sequence identified at both sides
of the Bari1 cluster in the h39 region of the chromosome 2 of D. melanogaster. The global sequence
alignment and the dot-plot comparison are shown.
Table 2. The distribution of the Doc5 transposon in the D. melanogaster genome
Subject
Accession Start End Chromosome % Identity Alignment
Length Evalue Bit Score Chromosome
Map Position
NT_033779.5 23,430,152 23,429,509 2L 85.891 645 0 643 h35–36
NT_033779.5 23,037,532 23,037,717 2L 91.237 194 2.03 ×10−67 257 h35–36
NW_001845128.1 3990 4435 2CEN 100 446 0 824 deep het
NW_001845128.1 3875 3990 2CEN 100 116 1.24 ×10−54 215 deep het
NW_001844967.1 11,017 10,572 2CEN 100 446 0 824 deep het
NW_001844967.1 11,132 11,017 2CEN 100 116 1.24 ×10−54 215 deep het
NW_007931075.1 7023 6491 2CEN 82.655 565 2.43 ×10−121 436 deep het
NW_007931075.1 9914 9382 2CEN 82.655 565 2.43 ×10−121 436 deep het
NT_033778.4 396,636 397,234 2R 99.332 599 0 1083 h41–h44
NT_033778.4 165,422 164,833 2R 95.326 599 0 942 h41–h44
NT_033778.4 74,075 74,637 2R 92.833 586 0 824 h41–h44
NT_033778.4 298,134 297,689 2R 100 446 0 824 h41–h44
NT_033778.4 872,342 871,810 2R 82.655 565 2.43 ×10−121 436 h41–h44
NT_033778.4 875,233 874,701 2R 82.655 565 2.43 ×10−121 436 h41–h44
NT_033778.4 1,413,093 1,413,384 2R 91.333 300 2.47 ×10−111 403 h45
NT_033778.4 3,518,328 3,518,018 2R 88.179 313 1.95 ×10−97 357 h41–h44
NT_033778.4 5,012,337 5,012,064 2R 82.818 291 4.43 ×10−59 230 h46
NT_033778.4 298,249 298,134 2R 100 116 1.24 ×10−54 215 h41-h44
NT_033778.4 5,012,596 5,012,448 2R 84.302 172 4.59 ×10−34 147 h46
NT_037436.4 24,877,579 24,878,162 3L 87.081 596 0.0 656 h49
NT_037436.4 24,914,416 24,913,834 3L 86.745 596 0.0 645 h49

Genes 2021,12, 1997 7 of 17
Table 2. Cont.
Subject
Accession Start End Chromosome % Identity Alignment
Length Evalue Bit Score Chromosome
Map Position
NT_037436.4 24,944,076 24,944,602 3L 87.199 539 2.93 ×10−170 599 h49
NT_037436.4 24,461,859 24,462,331 3L 85.443 474 6.71 ×10−127 455 h47
NT_037436.4 23,664,082 23,663,846 3L 94.583 240 9.00 ×10−101 368 80F9
NT_037436.4 23,663,844 23,663,639 3L 92.754 207 1.20 ×10−79 298 80F9
NT_037436.4 25,490,094 25,490,271 3L 94.382 178 2.02 ×10−72 274 h49–h50
NT_037436.4 27,913,043 27,913,159 3L 93.277 119 7.57 ×10−42 172 h51
NT_037436.4 24,502,780 24,502,891 3L 92.035 113 2.12 ×10−37 158 h48
NT_037436.4 27,912,886 27,913,038 3L 81.609 174 2.15 ×10−27 124 h51
NT_033777.3 646,928 646,337 3R 86.612 605 0.0 649 h54–h56
NT_033777.3 4,042,323 4,042,066 3R 94.961 258 6.86 ×10−112 405 81F
NT_033777.3 1,401,453 1,401,023 3R 83.991 431 1.50 ×10−103 377 h54–h56
NT_033777.3 4,050,664 4,050,455 3R 95.238 210 3.28 ×10−90 333 81F
NT_033777.3 3,992,575 3,992,365 3R 94.787 211 1.53 ×10−88 327 81F
NT_033777.3 4,039,954 4,039,769 3R 91.710 193 1.57 ×10−68 261 81F
NT_033777.3 2,453,123 2,453,400 3R 80.357 280 4.53 ×10−44 180 h56
NT_033777.3 2,453,399 2,453,508 3R 92.793 111 5.90 ×10−38 159 h56
NW_001845051.1 2554 2831 UNK 80.357 280 4.53 ×10−44 180 deep het
NW_001845051.1 2830 2934 UNK 89.189 111 1.29 ×10−29 132 deep het
The Doc5 sequence (596 bp) was used as a query in BlastN analyses against the D. melanogaster reference genome (Release 6). The
approximate map positions in the rightmost column were inferred by comparison with the data in [
12
]. Only alignments longer than
100 bases are shown. Deep het: deep heterochromatin. UNK: unknown map position.
Genes 2021, 12, x FOR PEER REVIEW 7 of 16
NT_033778.4 298,249 298,134 2R 100 116 1.24 × 10
−54
215 h41-h44
NT_033778.4 5,012,596 5,012,448 2R 84.302 172 4.59 × 10
−34
147 h46
NT_037436.4 24,877,579 24,878,162 3L 87.081 596 0.0 656 h49
NT_037436.4 24,914,416 24,913,834 3L 86.745 596 0.0 645 h49
NT_037436.4 24,944,076 24,944,602 3L 87.199 539 2.93 × 10
−170
599 h49
NT_037436.4 24,461,859 24,462,331 3L 85.443 474 6.71 × 10
−127
455 h47
NT_037436.4 23,664,082 23,663,846 3L 94.583 240 9.00 × 10
−101
368 80F9
NT_037436.4 23,663,844 23,663,639 3L 92.754 207 1.20 × 10
−79
298 80F9
NT_037436.4 25,490,094 25,490,271 3L 94.382 178 2.02 × 10
−72
274 h49–h50
NT_037436.4 27,913,043 27,913,159 3L 93.277 119 7.57 × 10
−42
172 h51
NT_037436.4 24,502,780 24,502,891 3L 92.035 113 2.12 × 10
−37
158 h48
NT_037436.4 27,912,886 27,913,038 3L 81.609 174 2.15 × 10
−27
124 h51
NT_033777.3 646,928 646,337 3R 86.612 605 0.0 649 h54–h56
NT_033777.3 4,042,323 4,042,066 3R 94.961 258 6.86 × 10
−112
405 81F
NT_033777.3 1,401,453 1,401,023 3R 83.991 431 1.50 × 10
−103
377 h54–h56
NT_033777.3 4,050,664 4,050,455 3R 95.238 210 3.28 × 10
−90
333 81F
NT_033777.3 3,992,575 3,992,365 3R 94.787 211 1.53 × 10
−88
327 81F
NT_033777.3 4,039,954 4,039,769 3R 91.710 193 1.57 × 10
−68
261 81F
NT_033777.3 2,453,123 2,453,400 3R 80.357 280 4.53 × 10
−44
180 h56
NT_033777.3 2,453,399 2,453,508 3R 92.793 111 5.90 × 10
−38
159 h56
NW_001845051.1 2554 2831 UNK 80.357 280 4.53 × 10
−44
180 deep het
NW_001845051.1 2830 2934 UNK 89.189 111 1.29 × 10
−29
132 deep het
The heterochromatic localization of the Doc5 element is also a conserved feature in
closely related species of the melanogaster complex, such as D. simulans and D. sechellia,
as demonstrated by the results of FISH experiments on polytene chromosomes (Figure 2).
Figure 2. The distribution of the Doc5 transposon was analyzed by FISH in the genome of D. sechellia
(left panel) and D. simulans (right panel), two species closely related to D. melanogaster. The Doc5
fragment cloned from the h39 region (596bp sequence) was used as probe. Arrowheads point to the
chromocenter.
The hybridization signals in the chromocenter and at the eu-heterochromatin transi-
tion on the chromosome arms (Figure 2) clearly highlight a heterochromatin-specific pat-
tern of Doc5, which is conserved in D. simulans and D. sechellia. The positional conserva-
tion of a transposon relic might indicate its possible functional or structural role, such as
the determination of the chromatin identity domains or the implication in transcriptional
processes.
Figure 2.
The distribution of the Doc5 transposon was analyzed by FISH in the genome of D. sechellia
(
left panel
) and D. simulans (
right panel
), two species closely related to D. melanogaster. The Doc5
fragment cloned from the h39 region (596bp sequence) was used as probe. Arrowheads point to
the chromocenter.
The hybridization signals in the chromocenter and at the eu-heterochromatin transi-
tion on the chromosome arms (Figure 2) clearly highlight a heterochromatin-specific pattern
of Doc5, which is conserved in D. simulans and D. sechellia. The positional conservation of a
transposon relic might indicate its possible functional or structural role, such as the deter-
mination of the chromatin identity domains or the implication in
transcriptional processes
.
The evolutionary conservation of the heterochromatic pattern and the high degree
of sequence identity of the Doc5 fragment duplicated at both sides of the Bari1 cluster
prompted us to hypothesize a possible structural role of the Doc5 sequence both in the
heterochromatin of D. melanogaster and in the identity of the h39. It was previously

Genes 2021,12, 1997 8 of 17
suggested that the preservation of a repetitive non-coding DNA sequence, especially in the
heterochromatin, could be promoted with the aid of stabilizing binding proteins [
41
], such
as chromatin proteins. To test this hypothesis, we performed a One-Hybrid System assay
aimed at the identification of proteins that potentially interact with the Doc5 fragment.
The double selection method (i.e., His prototrophy and positivity to the
β
-galactosidase
test) applied to identify positive clones ensures that the false positive rate is minimized.
Twenty-four positive clones, selected on selective media lacking histidine, were further
tested with the
β
-galactosidase activity (Table S1). Many of these clones turned rapidly
blue upon
β
-galactosidase testing (3–5 h). However, a large fraction (46%) of such clones
matched to Rpl22 transcripts after Sanger sequencing and BLASTN analysis. Based on the
fastness of color turning (the smallest the better) and the relative abundance of positive
clones, we chose Rpl22 as the candidate for further investigations.
The positive clones obtained from the first round of screening were further validated
by independent transformation of the isolated plasmids into the bait-containing yeast
strains (i.e., yeast strains containing the Doc5-His and Doc5-lacZ cassette, data not shown)
to confirm the bait–pray interaction.
To further solidify this result, we assayed the Rpl22–Doc5 interaction
in vitro
. The
Rpl22 protein was expressed and purified in E. coli and used for
in vitro
binding assays in
order to test its ability to bind an end-labeled Doc5 fragment (see Materials and Methods).
As can be observed in Figure 3(lanes 3–5), increasing amounts of purified protein led
to a slower migration in a polyacrylamide gel, suggesting the formation of progressively
slower DNA–protein complexes. In our hands, 3
µ
g of the protein extract led to the
formation of the slowest DNA–protein complex. This pattern could be explained by
either the presence of multiple binding sites in the target or by the possible formation of
multimeric protein complexes that bind the target fragment.
Genes 2021, 12, x FOR PEER REVIEW 8 of 16
The evolutionary conservation of the heterochromatic pattern and the high degree of
sequence identity of the Doc5 fragment duplicated at both sides of the Bari1 cluster
prompted us to hypothesize a possible structural role of the Doc5 sequence both in the
heterochromatin of D. melanogaster and in the identity of the h39. It was previously sug-
gested that the preservation of a repetitive non-coding DNA sequence, especially in the
heterochromatin, could be promoted with the aid of stabilizing binding proteins [41], such
as chromatin proteins. To test this hypothesis, we performed a One-Hybrid System assay
aimed at the identification of proteins that potentially interact with the Doc5 fragment.
The double selection method (i.e., His prototrophy and positivity to the β-galacto-
sidase test) applied to identify positive clones ensures that the false positive rate is mini-
mized.
Twenty-four positive clones, selected on selective media lacking histidine, were fur-
ther tested with the β-galactosidase activity (Table S1). Many of these clones turned rap-
idly blue upon β-galactosidase testing (3–5 h). However, a large fraction (46%) of such
clones matched to Rpl22 transcripts after Sanger sequencing and BLASTN analysis. Based
on the fastness of color turning (the smallest the better) and the relative abundance of
positive clones, we chose Rpl22 as the candidate for further investigations.
The positive clones obtained from the first round of screening were further validated
by independent transformation of the isolated plasmids into the bait-containing yeast
strains (i.e., yeast strains containing the Doc5-His and Doc5-lacZ cassette, data not shown)
to confirm the bait–pray interaction.
To further solidify this result, we assayed the Rpl22–Doc5 interaction in vitro. The
Rpl22 protein was expressed and purified in E. coli and used for in vitro binding assays in
order to test its ability to bind an end-labeled Doc5 fragment (see Materials and Methods).
As can be observed in Figure 3 (lanes 3–5), increasing amounts of purified protein
led to a slower migration in a polyacrylamide gel, suggesting the formation of progres-
sively slower DNA–protein complexes. In our hands, 3 μg of the protein extract led to the
formation of the slowest DNA–protein complex. This pattern could be explained by either
the presence of multiple binding sites in the target or by the possible formation of multi-
meric protein complexes that bind the target fragment.
Figure 3. The binding of Rpl22 to Doc5. The amount of labeled fragment (Doc5 *, Figure 3) in each
lane is 3 ng. The amounts of unlabeled specific competitor (ng of Doc5), Rpl22 (μg), and unlabeled
non-specific competitor (ng of linearized pUC19) are indicated in the figure legend under the re-
spective lanes. Increasing amounts of purified Rpl22 protein (lanes 3–5) and non-specific (lanes 6–
9) and specific (lanes 10–11) competitors are indicated on the top by triangles. A negative control
(lane 2) was performed following the incubation of the Doc5-labeled probe with 3 μg of non-induced
E. coli (BL21 strain) lysate (indicated with B).
Figure 3.
The binding of Rpl22 to Doc5. The amount of labeled fragment (Doc5 *, Figure 3) in
each lane is 3 ng. The amounts of unlabeled specific competitor (ng of Doc5), Rpl22 (
µ
g), and
unlabeled non-specific competitor (ng of linearized pUC19) are indicated in the figure legend under
the respective lanes. Increasing amounts of purified Rpl22 protein (lanes 3–5) and non-specific (lanes
6–9) and specific (lanes 10–11) competitors are indicated on the top by triangles. A negative control
(lane 2) was performed following the incubation of the Doc5-labeled probe with 3
µ
g of non-induced
E. coli (BL21 strain) lysate (indicated with B). The labeled fragments are indicated with an asterisk (*).
The observed protein binding is specific and reversible, as demonstrated by the
competition assays in Figure 3. While a 200-fold amount of unspecific competitor is not
sufficient to disrupt the Rpl22–Doc5 interaction (Figure 3, lanes 6–9), a 30-fold amount
of target fragment completely disrupts the observed DNA–protein binding (Figure 3,
lanes 10–11
). Additional controls to assess the specificity of the binding were performed

Genes 2021,12, 1997 9 of 17
using either an unrelated DNA fragment, or using a different non-specific competitor DNA
(Figure S1).
We next investigated whether the two domains of Rpl22 could differentially contribute
to the observed DNA–protein interaction. The H1-H5 domain and the ribosomal domain
were independently tested in EMSA assays for their ability to interact with Doc5. As
can be observed in Figure 4, only the H1-H5 domain retains the ability to bind the Doc5
fragment tested (Figure 4, lane 3), whereas the ribosomal domain does not (Figure 4,
lane 2
)
if compared to the binding observed for the wild-type Rpl22 protein (Figure 4, lane 4).
Similar to what observed for the wild-type protein (Figure 3, lanes 3–5), the H1–H5 domain
interacts with the Doc5 sequence in a dose-dependent manner (Figure 4B).
Genes 2021, 12, x FOR PEER REVIEW 9 of 16
The observed protein binding is specific and reversible, as demonstrated by the com-
petition assays in Figure 3. While a 200-fold amount of unspecific competitor is not suffi-
cient to disrupt the Rpl22–Doc5 interaction (Figure 3, lanes 6–9), a 30-fold amount of target
fragment completely disrupts the observed DNA–protein binding (Figure 3, lanes 10–11).
Additional controls to assess the specificity of the binding were performed using either
an unrelated DNA fragment, or using a different non-specific competitor DNA (Figure
S1).
We next investigated whether the two domains of Rpl22 could differentially contrib-
ute to the observed DNA–protein interaction. The H1-H5 domain and the ribosomal do-
main were independently tested in EMSA assays for their ability to interact with Doc5. As
can be observed in Figure 4, only the H1-H5 domain retains the ability to bind the Doc5
fragment tested (Figure 4, lane 3), whereas the ribosomal domain does not (Figure 4, lane
2) if compared to the binding observed for the wild-type Rpl22 protein (Figure 4, lane 4).
Similar to what observed for the wild-type protein (Figure 3, lanes 3–5), the H1–H5 do-
main interacts with the Doc5 sequence in a dose-dependent manner (Figure 4B).
Figure 4. Dissection of the DNA-binding domain of Rpl22 in vitro. (A) EMSA analysis of the ribosomal and the histone-
like domains of Rpl22. (B) EMSA analysis of the histone-like domain. A total of 3 μg of the Rpl22 (WT) and 1.5 μg of the
H1-H5 and ribosomal domains were used to maintain the unaltered DNA:protein molar ratio. A schematic representation
of the two main domains of Rpl22 protein is depicted at the top of the figure. Asterisk indicates that the fragment is
labelled.
To further investigate the possible role of Rpl22 in the chromatin dynamics, we tested
the Rpl22 protein localization in both D. melanogaster cultured cells, in order to check
whether the protein co-localizes with chromosomes. We performed immunofluorescence
localization of the native Rpl22 protein on polytene chromosomes of the Oregon-R wild-
type strain and inn cultured S2R+ cells, using a polyclonal antibody raised against the
Rpl22 protein.
The results obtained (Figure 5) clearly show that Rpl22 localizes to the nuclei, with a
marked nucleolar localization that has been further confirmed by co-localization with the
nucleolar marker fibrillarin, (Figure S2) both in salivary gland cells (Figure 5A) and in
cultured cells (Figure 5B), without any additional evidence of localization to chromatin.
Figure 4.
Dissection of the DNA-binding domain of Rpl22
in vitro
. Labeled fragments are indicated with an asterisk (*).
(
A
) EMSA analysis of the ribosomal and the histone-like domains of Rpl22. (
B
) EMSA analysis of the histone-like domain.
A total of 3
µ
g of the Rpl22 (WT) and 1.5
µ
g of the H1-H5 and ribosomal domains were used to maintain the unaltered
DNA:protein molar ratio. A schematic representation of the two main domains of Rpl22 protein is depicted at the top of the
figure. Asterisk indicates that the fragment is labelled.
To further investigate the possible role of Rpl22 in the chromatin dynamics, we tested
the Rpl22 protein localization in both D. melanogaster cultured cells, in order to check
whether the protein co-localizes with chromosomes. We performed immunofluorescence
localization of the native Rpl22 protein on polytene chromosomes of the Oregon-R wild-
type strain and inn cultured S2R+ cells, using a polyclonal antibody raised against the
Rpl22 protein.
The results obtained (Figure 5) clearly show that Rpl22 localizes to the nuclei, with
a marked nucleolar localization that has been further confirmed by co-localization with
the nucleolar marker fibrillarin, (Figure S2) both in salivary gland cells (Figure 5A) and in
cultured cells (Figure 5B), without any additional evidence of localization to chromatin.
In silico prediction of the nuclear localization of Rpl22 using cNLS Mapper [
38
]
suggests its nuclear localization, with the best scoring NLS signal (score 7/7) mapped at
position 234. A similar search, using NucPred [
39
] as an alternative algorithm, returned
the sequence GKGQKKKK (position 181, score 0.28; a 0.30 threshold corresponds to 77%
sensitivity and 55% specificity).

Genes 2021,12, 1997 10 of 17
Genes 2021, 12, x FOR PEER REVIEW 10 of 16
Figure 5. Pattern of subcellular immunolocalization of Rpl22 in D. melanogaster salivary gland nuclei (A) and in cultured
S2R+ cells (B). White arrowheads point to nucleoli. A magnified detail of the nucleolar co-localization is reported in the
inset. Additional details on the localization of Rpl22 to nucleoli are given in Figure S2.
In silico prediction of the nuclear localization of Rpl22 using cNLS Mapper [38] sug-
gests its nuclear localization, with the best scoring NLS signal (score 7/7) mapped at posi-
tion 234. A similar search, using NucPred [39] as an alternative algorithm, returned the
sequence GKGQKKKK (position 181, score 0.28; a 0.30 threshold corresponds to 77% sen-
sitivity and 55% specificity).
In the absence of additional experimental evidence, the possible role of Rpl22 in the
heterochromatin can be inferred from interactomic data obtained in previously published
works. Out of the ninety-one RpL22-interacting proteins that are annotated in FlyBase, 13
are non-RPs. Notably, 12 out of the 13 interacting proteins are not directly linked with the
translational machinery.
Rpl22 interacts with protein involved in heterochromatin organization (vig and vig2
[42,43]), piRNA biogenesis (Fmr1 and its associated miRNA, bantam [44]), and transcrip-
tional repression (Ago1 and Ago2 [42]) (Table 3). Such interactions further suggest the
involvement of Rpl22 in chromatin determination and transcriptional pathways, support-
ing our hypothesis.
Figure 5.
Pattern of subcellular immunolocalization of Rpl22 in D. melanogaster salivary gland nuclei (
A
) and in cultured
S2R+ cells (
B
). White arrowheads point to nucleoli. A magnified detail of the nucleolar co-localization is reported in the
inset. Additional details on the localization of Rpl22 to nucleoli are given in Figure S2.
In the absence of additional experimental evidence, the possible role of Rpl22 in the
heterochromatin can be inferred from interactomic data obtained in previously published
works. Out of the ninety-one RpL22-interacting proteins that are annotated in FlyBase, 13
are non-RPs. Notably, 12 out of the 13 interacting proteins are not directly linked with the
translational machinery.
Rpl22 interacts with protein involved in heterochromatin organization (vig and
vig2 [
42
,
43
]), piRNA biogenesis (Fmr1 and its associated miRNA, bantam [
44
]), and tran-
scriptional repression (Ago1 and Ago2 [
42
]) (reported in Table 3). Such interactions further
suggest the involvement of Rpl22 in chromatin determination and transcriptional pathways,
supporting our hypothesis.
Table 3.
Rpl22 interacting proteins involved in heterochromatin functions. Information retrieved
from Flybase (last accessed August 2021).
Gene Name FlyBase ID Function Inferred by Reference
vig FBgn0024183 Heterochromatin organization Co-IP [42]
AGO1 FBgn0262739 transcriptional repression Co-IP [42]
AGO2 FBgn0087035 transcriptional repression Co-IP [42]

Genes 2021,12, 1997 11 of 17
Table 3. Cont.
Gene Name FlyBase ID Function Inferred by Reference
vig2 FBgn0046214 Heterochromatin organization Mass-spec [43]
Fmr1 FBgn0028734 piRNA biogenesis Co-IP [42]
ban FBgn0262451 piRNA biogenesis Co-IP [42]
esi2 FBgn0285992 Unknown Co-IP [42]
smt3 FBgn0264922 mitosis Co-IP [31]
4. Discussion
The stabilization of large chromosomal domains containing extended repeat blocks
essentially depends on the chromatin architecture that wraps these loci. Both the Encode [
5
]
and modEncode [
45
] projects have had a leading role in the determination of the genome-
wide chromatin status in H. sapiens and model organisms, respectively. The outcome
of these huge projects led to the birth of epigenomics that aims to link cell-type-specific
gene expression to chromatin structure. The specific features of chromatin domains are
also of critical importance for genome evolution since the propensity of certain loci to
be converted and relocated from the euchromatin to the heterochromatin is probably
determined by the ancestral epigenetic marks [
46
]. For this reason, profound knowledge of
chromatin dynamics is fundamental in the determination of the evolutionary trajectories
that chromosomes follow.
However, chromatin is a highly dynamic biological entity, and for this reason, it is
difficult to provide a definitive and exhaustive description. Unbiased approaches, i.e., not
focused on a particular developmental stage or specific tissue, allow for a near-to-complete
characterization of chromatin-associated proteins. It follows that the elucidation of the
changing state of chromatin in the most diverse cellular types is of particular importance
toward the complete understanding of physiological and pathological conditions [47].
Here, we report that a ribosomal protein binds the Doc5 transposon, a non-autonomous
TE family enriched in the heterochromatin of D. melanogaster and closely related species [
48
],
providing
in vitro
experimental evidence for a functional interaction of Rpl22 with DNA,
and possibly to chromosome and chromatin. In Drosophila, the direct binding of protein
to TEs, especially involving retrotransposons, has been previously reported [
49
–
51
]. In a
yeast one-hybrid assay, we probed a D. melanogaster expression library with Doc5 as bait
and found Rpl22 as the best candidate interacting protein. We have further validated the
DNA–protein interaction with a series of EMSA experiments that confirmed the results of
the experiments in yeast. We further demonstrated that the NH-terminal domain (H1–H5
domain) of the protein is both necessary and sufficient to bind DNA. Furthermore, the
assays performed
in vitro
show that the Doc5–Rpl22 interaction depends on the amount of
protein input. We cannot dismiss the hypothesis that this behavior could depend both on
the presence of multiple binding sites on the target (which we have not investigated), and
on the ability of Rpl22 to multimerize or to form homogeneous aggregates. In addition, the
net charge density of the expressed H1-H5 domain is greater than that of the wild-type
Rpl22 protein (27.14/15.8 KDa vs. 36.51/30.6 KDa, respectively, at pH = 7), which can
account for the increased shift of the H1-H5/Doc5 complex if compared to the wild-type
Rpl22/Doc5 complex (Figure 4).
What is the relevance of our findings? Our results let us hypothesize that Rpl22 could
have a potential role in the organization of chromatin, possibly in heterochromatin, and
this hypothesis is supported by several studies reporting that RPs are linked to biological
processes occurring in the nucleus [
52
]. RPs have been found associated at transcription
sites in Drosophila polytene chromosomes. This unexpected finding suggested that ribo-
somal subunits could be associated with nascent mRNAs [
53
]. An additional study in
Saccharomyces cerevisiae showed that RPs bind to noncoding RNA genes, suggesting that
the RPs–RNA association might be independent of the translatability of the transcript and

Genes 2021,12, 1997 12 of 17
might involve free RPs that are not assembled into ribosomes [
54
]. Several other examples
of RPs with extra ribosomal functions at transcription sites have been reported to date.
Some RPs auto-regulate their expression by affecting translation, splicing, or transcription
by interacting with their mRNA, or promoter [
55
–
57
]. RPs are also able to interact with
transcription factors at the promoters of genes. RpL11 binds the oncoprotein c-MYC at
the promoter of c-MYC target genes [
58
,
59
], RpS3 is a subunit of the NF-
κ
B DNA-binding
complex involved in chromatin binding and transcription regulation of specific genes [
60
].
RpS3 phosphorylation at serine 209 by IKKb is crucial for RPS3 nuclear localization in
response to activating stimuli [61].
Rpl22 is a ribosomal protein with a prevailing cytoplasmic localization. Past-published
reports claimed that Rpl22 also localizes to the nucleus of Drosophila cells. Ni and collabora-
tors [
62
] demonstrated that Rpl22 expressed at endogenous levels localizes in the nucleus
of Drosophila Kc (embryo-derived) and cl-8 (derived from imaginal discs) cell cultures,
and it is associated with chromatin, resulting in gene suppression. Immunofluorescent
staining and chromatin immunoprecipitation (ChIP) analyses demonstrated that RpL22
and H1 are both associated with condensed chromatin. In the same study, it was demon-
strated that the overexpression of RpL22 caused the transcriptional repression of two-thirds
of the genes suppressed by histone H1. By contrast, RpL22 depletion caused the up-
regulation of the transcription of several tested genes, supporting a role for RpL22 as
transcriptional repressor [
62
]. These observations imply the involvement of Rpl22 in global
transcriptional processes.
However, Rpl22 has not been previously identified in surveys aimed at the identifica-
tion of chromatin structure. This can be due to an experimental bias when searching histone
modifications [
63
]. On the other hand, unbiased studies have been focused on euchromatic
genomic regions only [
64
]. Conversely, our approach was based on the search of proteins
that interact with a heterochromatic sequence, and our results support a role of Rpl22 in
the chromatin. To what extent Rpl22 could participate in the determination of chromatin
domains remains to be determined. Another potential implication of our findings concerns
the possible role of the non-autonomous Doc5 transposon in the D. melanogaster genome.
Non-autonomous TEs often acquire new functions in complex genomes, over evolutionary
time. Many examples of evolutionarily inactive TEs that have been co-opted, exapted, or
domesticated are described in the scientific literature [65,66].
It has been demonstrated that Doc5 is under the control of the piRNA pathway [
67
,
68
].
Since no potentially active Doc5 copies are found, these findings suggest that the short
RNAs generated from Doc5 could have alternative roles in the regulation of gene expression,
or alternatively in the regulation of the transposition of other, unrelated, TEs.
Alternatively, Doc5 could mark a chromatin domain with a structural function that
prevents the excessive expansion of the Bari1 cluster. This hypothesis could be extended
to other species-specific heterochromatic repeats, since the Bari1 cluster is unique to the
D. melanogaster species, while Doc5 is present in the genome of sibling species [17].
In contrast with previously reported results, we were not able to demonstrate/reproduce
a pan-nuclear localization of Rpl22 in S2R+ cells. Our experiments only revealed a nucleolar
localization of the protein, without any detectable association to chromatin. This contrasting
result could be explained considering the limitations of the immunolocalization technique,
which would not allow for the detection of a small amount of chromatin proteins. Moreover,
the differences between the cell lines used in our experimental setup (S2R+) and in previous
studies (Kc) should be taken into account. Kc are male derived, while S2R+ derive from
females. Kc have a plasmatocyte-like phenotype, while S2R+ combines properties of
plasmatocytes and crystal cells [
69
]. Finally, Kc and S2R+ have different ploidy, since Kc
are approximately 4n, while S2R+ are 2.5n [
70
]. We can, therefore, hypothesize that the
nuclear localization and the association to chromatin is cell-type dependent. Consequently,
we cannot dismiss the fact that, in S2R+ cells, Rpl22 can also enter the nucleus under
particular experimental conditions. An additional limitation of our study is the lack of
confocal microscopy analyses, which grants a powerful resolution at the subcellular level.

Genes 2021,12, 1997 13 of 17
Similarly, we did not detect Rpl22 signals on the polytene chromosome arms, nor
in the chromocenter. The limitation in terms of resolution using the polytene chromo-
some to assess the presence of DNA–protein interactions in heterochromatin is due to the
under-replicated nature of the chromocenter [
71
,
72
]. Moreover, it has been demonstrated
that Rpl22 is subjected to post-translational modifications in testis [
31
]. SUMOylation,
phosphorylation, and possibly other unexplored post-translational modifications could
also affect the Rpl22 localization and its ability to be engaged in additional functions,
other than translation. Post-translationally modified Rpl22 could potentially exit from the
nucleolus and associate to chromatin in particular, unexplored, physiological conditions
or in response to environmental stresses. This change in localization could be elicited by
protein post-translational modification, as demonstrated in previous studies involving
Rpl22 [31].
Despite the lack of localization to chromosomes and chromatin, several additional
observations support the hypothesis of an involvement of Rpl22 in chromatin dynamics.
There are 12 out of 91 Rpl22-interacting proteins that suggest its involvement in chromatin-
related processes. Furthermore, RpL22 has been identified as one of the two hundred genes
required for mitotic spindle assembly in Drosophila S2 cells in an RNAi screen [
73
], and the
down-regulation of the RpL22 gene also results in aberrantly short, monopolar spindles in
S2 cells. These data together with the demonstration of the DNA binding ability of Rpl22
presented in this paper, offer a new perspective of how Rpl22 could participate in chromatin
dynamics, at least under specific conditions that has yet to be determined. Additionally,
previous genome-wide ChIP-on-chip analysis in the fission yeast S. pombe revealed the
presence of ribosomal protein complexes at transcription sites with unexpected peaks at
centromeres, raising the intriguing hypothesis that RP complexes are involved in tRNA
biogenesis and possibly centromere functions [57].
5. Conclusions
We have presented
in vitro
evidence of the interaction between a typical heterochro-
matic sequence and a ribosomal protein in D. melanogaster. However, experiments
in vivo
do not confirm the results of experiments
in vitro
, suggesting that further investigation is
needed to reveal the physiological role of Rpl22 in the context of the chromosome structure.
While further studies are needed to understand if the Doc5 element has been co-opted
to absolve further functions in the heterochromatin, several suggestive hypotheses could
be proposed. Doc5 could act as a bidirectional promoter that allows for the transcription of
the Bari1 cluster in order to activate the piRNA-mediated repression of the transposition.
In this hypothesis, the ribosomal protein Rpl22 could help in the transcriptional activation
from this promoter [
74
] or in the stabilization of non-coding RNAs [
54
]. Considering that
the Bari1 elements tested so far are transpositionally active [
20
,
21
,
75
] and are capable of
autonomous transcription [
76
,
77
], this hypothesis could at least partially explain the low
transposition activity observed in D. melanogaster laboratory strains [
20
]. In a companion
paper, Minervini et al. (Minervini et al. submitted to Genes) also demonstrated that Rpl22
binds
in vitro
to a transposable element-derived consensus sequence. This observation
leads to the hypothesis that ribosomal proteins could be also involved in controlling
the activity of TEs in Drosophila, not only at the translation level [
78
] but also at the
transcriptional level.

Genes 2021,12, 1997 14 of 17
Supplementary Materials:
The following are available online at https://www.mdpi.com/article/
10.3390/genes12121997/s1, Table S1: List of the yeast clones tested in the
β
-Galactosidase assay.
Supplementary Figure S1:
In vitro
assays (EMSA) suggesting the specificity of the Doc5/Rpl22
binding. Supplementary Figure S2: (
A
). Rpl22 co-localizes with fibrillarin in S2R+ cells. From the left
to the right: DAPI, anti-fibrillarin, anti-Rpl22, merged signals. Signal pseudo-coloring in the merged
image is as follows. DAPI: blue; Fibrillarin: red; Rpl22: green. The arrowhead in the merged image
point to the nucleolus. A magnified detail of the nucleolar co-localization is reported in the inset. (
B
).
Rpl22 co-localizes with fibrillarin in polytene nuclei. From the left to the right: DAPI, anti-fibrillarin,
anti-Rpl22, merged signals. Signal pseudo-coloring in the merged image is as follows. DAPI: blue;
Fibrillarin: green; Rpl22: red. Arrowheads in the merged image point to nucleoli. Table S1. List of the
yeast clones tested in the Galactosidase assay. Clones carrying Rpl22 sequences are reported in bold.
Author Contributions:
Conceptualization, R.M.M.; One Hybrid Experiments, C.F.M., R.M.M.; im-
munofluorescence experiments, M.F.B.; FISH experiments on polytene chromosomes, R.M. and
R.M.M.; in silico analyses, A.P.; supervised the project, L.V. and R.M.M.; writing—original draft
preparation, C.F.M., M.F.B., L.V., R.M.M.; writing—review and editing, C.F.M., M.F.B., L.V., R.M.M.
All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments:
A.P. is supported by a grant from Regione Puglia “Research for Innovation
(REFIN)”-POR PUGLIA FESR-FSE 2014/2020. Codice Pratica: B39303C8.
Conflicts of Interest: The authors declare no conflict of interest.
References
1. Kornberg, R.D. Chromatin Structure: A Repeating Unit of Histones and DNA. Science 1974,184, 868. [CrossRef] [PubMed]
2. Lindsay, S. Chromatin control of gene expression: The simplest model. Biophys. J. 2007,92, 1113. [CrossRef]
3.
Schmitt, A.D.; Hu, M.; Ren, B. Genome-wide mapping and analysis of chromosome architecture. Nat. Rev. Mol. Cell Biol.
2016
,17,
743–755. [CrossRef] [PubMed]
4.
Simpson, R.T. Structure of the chromatosome, a chromatin particle containing 160 base pairs of DNA and all the histones.
Biochemistry 1978,17, 5524–5531. [CrossRef]
5. Consortium, E.P. An integrated encyclopedia of DNA elements in the human genome. Nature 2012,489, 57–74. [CrossRef]
6.
Chen, Z.X.; Sturgill, D.; Qu, J.; Jiang, H.; Park, S.; Boley, N.; Suzuki, A.M.; Fletcher, A.R.; Plachetzki, D.C.;
FitzGerald, P.C.; et al
.
Comparative validation of the D. melanogaster modENCODE transcriptome annotation. Genome Res.
2014
,24, 1209–1223.
[CrossRef] [PubMed]
7. Arunkumar, G.; Melters, D.P. Centromeric Transcription: A Conserved Swiss-Army Knife. Genes 2020,11, 911. [CrossRef]
8.
Tachiwana, H.; Yamamoto, T.; Saitoh, N. Gene regulation by non-coding RNAs in the 3D genome architecture. Curr. Opin. Genet.
Dev. 2020,61, 69–74. [CrossRef] [PubMed]
9. Barral, A.; Déjardin, J. Telomeric Chromatin and TERRA. J. Mol. Biol. 2020,432, 4244–4256. [CrossRef]
10.
Zeng, C.; Fukunaga, T.; Hamada, M. Identification and analysis of ribosome-associated lncRNAs using ribosome profiling data.
BMC Genom. 2018,19, 414. [CrossRef]
11.
Mélèse, T.; Xue, Z. The nucleolus: An organelle formed by the act of building a ribosome. Curr. Opin. Cell Biol.
1995
,7, 319–324.
[CrossRef]
12.
Marsano, R.M.; Giordano, E.; Messina, G.; Dimitri, P. A New Portrait of Constitutive Heterochromatin: Lessons from Drosophila
melanogaster.Trends Genet. 2019,35, 615–631. [CrossRef] [PubMed]
13.
Larracuente, A.M.; Presgraves, D.C. The selfish Segregation Distorter gene complex of Drosophila melanogaster.Genetics
2012
,192,
33–53. [CrossRef]
14.
Berloco, M.; Fanti, L.; Fau-Breiling, A.; Breiling, A.; Fau-Orlando, V.; Orlando, V.; Fau-Pimpinelli, S.; Pimpinelli, S. The maternal
effect gene, abnormal oocyte (abo), of Drosophila melanogaster encodes a specific negative regulator of histones. Proc. Natl. Acad.
Sci. USA 2001,98, 12126–12131. [CrossRef]
15.
Tritto, P.; Specchia, V.; Fanti, L.; Berloco, M.; D’Alessandro, R.; Pimpinelli, S.; Palumbo, G.; Pia Bozzetti, M. Structure, Regulation
and Evolution of the Crystal–Stellate System of Drosophila.Genetica 2003,117, 247–257. [CrossRef]
16.
Caizzi, R.; Caggese, C.; Pimpinelli, S. Bari-1, a new transposon-like family in Drosophila melanogaster with a unique heterochromatic
organization. Genetics 1993,133, 335–345. [CrossRef]

Genes 2021,12, 1997 15 of 17
17. Palazzo, A.; Lovero, D.; D’Addabbo, P.; Caizzi, R.; Marsano, R.M. Identification of Bari Transposons in 23 Sequenced Drosophila
Genomes Reveals Novel Structural Variants, MITEs and Horizontal Transfer. PLoS ONE 2016,11, e0156014. [CrossRef]
18.
McGurk, M.P.; Barbash, D.A. Double insertion of transposable elements provides a substrate for the evolution of satellite DNA.
Genome Res. 2018,28, 714–725. [CrossRef] [PubMed]
19.
Caggese, C.; Pimpinelli, S.; Barsanti, P.; Caizzi, R. The distribution of the transposable element Bari-1 in the Drosophila
melanogaster and Drosophila simulans genomes. Genetica 1995,96, 269–283. [CrossRef]
20.
Palazzo, A.; Marconi, S.; Specchia, V.; Bozzetti, M.P.; Ivics, Z.; Caizzi, R.; Marsano, R.M. Functional Characterization of the Bari1
Transposition System. PLoS ONE 2013,8, e79385. [CrossRef]
21.
Palazzo, A.; Moschetti, R.; Caizzi, R.; Marsano, R.M. The Drosophila mojavensis Bari3 transposon: Distribution and functional
characterization. Mob. DNA 2014,5, 21. [CrossRef]
22.
Sandler, L.; Hiraizumi, Y.; Fau-Sandler, I.; Sandler, I. Meiotic Drive in Natural Populations of Drosophila Melanogaster. I. the
Cytogenetic Basis of Segregation-Distortion. Genetics 1959,44, 233. [CrossRef] [PubMed]
23.
Pimpinelli, S.; Dimitri, P. Cytogenetic analysis of segregation distortion in Drosophila melanogaster: The cytological organization of
the Responder (Rsp) locus. Genetics 1989,121, 765–772. [CrossRef] [PubMed]
24.
Moschetti, R.; Palazzo, A.; Lorusso, P.; Viggiano, L.; Marsano, R.M. “What You Need, Baby, I Got It”: Transposable Elements as
Suppliers of Cis-Operating Sequences in Drosophila.Biology 2020,9, 25. [CrossRef] [PubMed]
25.
Marsano, R.M.; Milano, R.; Minervini, C.; Moschetti, R.; Caggese, C.; Barsanti, P.; Caizzi, R. Organization and possible origin of
the Bari-1 cluster in the heterochromatic h39 region of Drosophila melanogaster.Genetica 2003,117, 281–289. [CrossRef]
26.
Moschetti, R.; Caizzi, R.; Pimpinelli, S. Segregation distortion in Drosophila melanogaster: Genomic organization of Responder
sequences. Genetics 1996,144, 1365–1371. [CrossRef] [PubMed]
27.
Marsano, R.M.; Moschetti, R.; Barsanti, P.; Caggese, C.; Caizzi, R. A survey of the DNA sequences surrounding the Bari1 repeats
in the pericentromeric h39 region of Drosophila melanogaster.Gene 2003,307, 167–174. [CrossRef]
28.
Zhao, W.; Bidwai, A.P.; Glover, C.V.C. Interaction of casein kinase II with ribosomal protein L22 of Drosophila melanogaster.Biochem.
Biophys. Res. Commun. 2002,298, 60–66. [CrossRef]
29.
Bayona-Feliu, A.; Casas-Lamesa, A.; Reina, O.; Bernués, J.; Azorín, F. Linker histone H1 prevents R-loop accumulation and
genome instability in heterochromatin. Nat. Commun. 2017,8, 283. [CrossRef]
30.
Koyama, Y.; Katagiri, S.; Hanai, S.; Uchida, K.; Miwa, M. Poly(ADP-ribose) polymerase interacts with novel Drosophila ribosomal
proteins, L22 and L23a, with unique histone-like amino-terminal extensions. Gene 1999,226, 339–345. [CrossRef]
31.
Kearse, M.G.; Ireland, J.A.; Prem, S.M.; Chen, A.S.; Ware, V.C. RpL22e, but not RpL22e-like-PA, is SUMOylated and localizes to
the nucleoplasm of Drosophila meiotic spermatocytes. Nucleus 2013,4, 241–258. [CrossRef] [PubMed]
32.
Mageeney, C.M.; Ware, V.C. Specialized eRpL22 paralogue-specific ribosomes regulate specific mRNA translation in spermatoge-
nesis in Drosophila melanogaster.Mol. Biol. Cell 2019,30, 2240–2253. [CrossRef]
33. Gietz, R.D. Yeast transformation by the LiAc/SS carrier DNA/PEG method. Methods Mol. Biol. 2014,1205, 1–12.
34.
Colloms, S.D.; van Luenen, H.G.; Plasterk, R.H. DNA binding activities of the Caenorhabditis elegans Tc3 transposase. Nucleic
Acids Res. 1994,22, 5548–5554. [CrossRef]
35.
Marsano, R.M.; Moschetti, R.; Caggese, C.; Lanave, C.; Barsanti, P.; Caizzi, R. The complete Tirant transposable element in
Drosophila melanogaster shows a structural relationship with retrovirus-like retrotransposons. Gene 2000,247, 87–95. [CrossRef]
36.
James, T.C.; Eissenberg, J.C.; Craig, C.; Dietrich, V.; Hobson, A.; Elgin, S.C. Distribution patterns of HP1, a heterochromatin-
associated nonhistone chromosomal protein of Drosophila.Eur. J. Cell Biol. 1989,50, 170–180.
37.
Marck, C. ‘DNA Strider’: A ‘C’ program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of
computers. Nucleic Acids Res. 1988,16, 1829–1836. [CrossRef]
38.
Kosugi, S.; Hasebe, M.; Fau-Tomita, M.; Tomita, M.; Fau-Yanagawa, H.; Yanagawa, H. Systematic identification of cell cycle-
dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs. Proc. Natl. Acad. Sci. USA
2009
,106,
10171–10176. [CrossRef]
39.
Brameier, M.; Krings A Fau-MacCallum, R.M.; MacCallum, R.M. NucPred—Predicting nuclear localization of proteins. Bioinfor-
matics 2007,23, 1159–1160. [CrossRef] [PubMed]
40.
Coelho, P.A.; Nurminsky, D.; Hartl, D.; Sunkel, C.E. Identification of Porto-1, a new repeated sequence that localises close to the
centromere of chromosome 2 of Drosophila melanogaster.Chromosoma 1996,105, 211–222. [CrossRef] [PubMed]
41.
Csink, A.K.; Henikoff, S. Something from nothing: The evolution and utility of satellite repeats. Trends Genet.
1998
,14, 200–204.
[CrossRef]
42.
Zhou, R.; Hotta, I.; Denli, A.M.; Hong, P.; Perrimon, N.; Hannon, G.J. Comparative analysis of argonaute-dependent small RNA
pathways in Drosophila.Mol. Cell 2008,32, 592–599. [CrossRef]
43.
Anger, A.M.; Armache, J.-P.; Berninghausen, O.; Habeck, M.; Subklewe, M.; Wilson, D.N.; Beckmann, R. Structures of the human
and Drosophila 80S ribosome. Nature 2013,497, 80–85. [CrossRef] [PubMed]
44.
Yang, Y.; Xu, S.; Xia, L.; Wang, J.; Wen, S.; Jin, P.; Chen, D. The Bantam microRNA Is Associated with Drosophila Fragile X Mental
Retardation Protein and Regulates the Fate of Germline Stem Cells. PLoS Genet. 2009,5, e1000444. [CrossRef]
45.
Celniker, S.E.; Dillon, L.A.L.; Gerstein, M.B.; Gunsalus, K.C.; Henikoff, S.; Karpen, G.H.; Kellis, M.; Lai, E.C.; Lieb, J.D.;
MacAlpine, D.M.; et al. Unlocking the secrets of the genome. Nature 2009,459, 927–930. [CrossRef]

Genes 2021,12, 1997 16 of 17
46.
Caizzi, R.; Moschetti, R.; Piacentini, L.; Fanti, L.; Marsano, R.M.; Dimitri, P. Comparative Genomic Analyses Provide New Insights
into the Evolutionary Dynamics of Heterochromatin in Drosophila.PLoS Genet. 2016,12, e1006212. [CrossRef]
47. Tzika, E.; Dreker, T.; Imhof, A. Epigenetics and Metabolism in Health and Disease. Front. Genet. 2018,9, 361. [CrossRef]
48. Signor, S. Transposable elements in individual genotypes of Drosophila simulans. Ecol. Evol. 2020,10, 3402–3412. [CrossRef]
49.
Spana, C.; Harrison, D.A.; Corces, V.G. The Drosophila melanogaster suppressor of Hairy-wing protein binds to specific sequences
of the gypsy retrotransposon. Genes Dev. 1988,2, 1414–1423. [CrossRef] [PubMed]
50.
Georgiev, P.G.; Corces, V.G. The su(Hw) protein bound to gypsy sequences in one chromosome can repress enhancer-promoter
interactions in the paired gene located in the other homolog. Proc. Natl. Acad. Sci. USA 1995,92, 5184–5188. [CrossRef]
51.
Minervini, C.F.; Marsano, R.M.; Casieri, P.; Fanti, L.; Caizzi, R.; Pimpinelli, S.; Rocchi, M.; Viggiano, L. Heterochromatin protein 1
interacts with 5’UTR of transposable element ZAM in a sequence-specific fashion. Gene 2007,393, 1–10. [CrossRef] [PubMed]
52. Bhavsar, R.B.; Makley, L.N.; Tsonis, P.A. The other lives of ribosomal proteins. Hum. Genom. 2010,4, 327. [CrossRef] [PubMed]
53.
Brogna, S.; Sato, T.-A.; Rosbash, M. Ribosome Components Are Associated with Sites of Transcription. Mol. Cell
2002
,10, 93–104.
[CrossRef]
54.
Schroder, P.A.; Moore, M.J. Association of ribosomal proteins with nascent transcripts in S. cerevisiae. RNA
2005
,11, 1521–1529.
[CrossRef]
55. Wool, I.G. Extraribosomal functions of ribosomal proteins. Trends Biochem. Sci. 1996,21, 164–165. [CrossRef]
56.
Warner, J.R.; McIntosh, K.B. How Common Are Extraribosomal Functions of Ribosomal Proteins? Mol. Cell
2009
,34, 3–11.
[CrossRef]
57.
De, S.; Varsally, W.; Falciani, F.; Brogna, S. Ribosomal proteins’ association with transcription sites peaks at tRNA genes in
Schizosaccharomyces pombe. RNA 2011,17, 1713–1726. [CrossRef]
58.
Dai, M.-S.; Arnold, H.; Sun, X.-X.; Sears, R.; Lu, H. Inhibition of c-Myc activity by ribosomal protein L11. Embo J.
2007
,26,
3332–3345. [CrossRef]
59.
Dai, M.-S.; Sun, X.-X.; Lu, H. Ribosomal protein L11 associates with c-Myc at 5 S rRNA and tRNA genes and regulates their
expression. J. Biol. Chem. 2010,285, 12587–12594. [CrossRef]
60.
Wan, F.; Anderson, D.E.; Barnitz, R.A.; Snow, A.; Bidere, N.; Zheng, L.; Hegde, V.; Lam, L.T.; Staudt, L.M.; Levens, D.; et al.
Ribosomal Protein S3: A KH Domain Subunit in NF-
κ
B Complexes that Mediates Selective Gene Regulation. Cell
2007
,131,
927–939. [CrossRef] [PubMed]
61.
Wan, F.; Weaver, A.; Gao, X.; Bern, M.; Hardwidge, P.R.; Lenardo, M.J. IKK
β
phosphorylation regulates RPS3 nuclear translocation
and NF-
κ
B function during infection with Escherichia coli strain O157:H7. Nat. Immunol.
2011
,12, 335–343. [CrossRef] [PubMed]
62.
Ni, J.-Q.; Liu, L.-P.; Hess, D.; Rietdorf, J.; Sun, F.-L. Drosophila ribosomal proteins are associated with linker histone H1 and
suppress gene transcription. Genes Dev. 2006,20, 1959–1973. [CrossRef]
63.
Kharchenko, P.V.; Alekseyenko, A.A.; Schwartz, Y.B.; Minoda, A.; Riddle, N.C.; Ernst, J.; Sabo, P.J.; Larschan, E.;
Gorchakov, A.A.;
Gu, T.; et al. Comprehensive analysis of the chromatin landscape in Drosophila melanogaster.Nature
2011
,471, 480–485. [CrossRef]
64.
Bonnet, J.; Lindeboom, R.G.H.; Pokrovsky, D.; Stricker, G.; Çelik, M.H.; Rupp, R.A.W.; Gagneur, J.; Vermeulen, M.; Imhof, A.;
Müller, J. Quantification of Proteins and Histone Marks in Drosophila Embryos Reveals Stoichiometric Relationships Impacting
Chromatin Regulation. Dev. Cell 2019,51, 632–644.e6. [CrossRef] [PubMed]
65.
Sinzelle, L.; Izsvak, Z.; Ivics, Z. Molecular domestication of transposable elements: From detrimental parasites to useful host
genes. Cell Mol. Life Sci. 2009,66, 1073–1093. [CrossRef]
66.
Cosby, R.L.; Chang, N.C.; Feschotte, C. Host-transposon interactions: Conflict, cooperation, and cooption. Genes Dev.
2019
,33,
1098–1116. [CrossRef] [PubMed]
67.
Pane, A.; Jiang, P.; Zhao, D.Y.; Singh, M.; Schupbach, T. The Cutoff protein regulates piRNA cluster expression and piRNA
production in the Drosophila germline. EMBO J. 2011,30, 4601–4615. [CrossRef]
68.
Zhao, K.; Cheng, S.; Miao, N.; Xu, P.; Lu, X.; Zhang, Y.; Wang, M.; Ouyang, X.; Yuan, X.; Liu, W.; et al. A Pandas complex adapted
for piRNA-guided transcriptional silencing and heterochromatin formation. Nat. Cell Biol. 2019,21, 1261–1272. [CrossRef]
69.
Cherbas, L.; Willingham, A.; Zhang, D.; Yang, L.; Zou, Y.; Eads, B.D.; Carlson, J.W.; Landolin, J.M.; Kapranov, P.; Dumais, J.; et al.
The transcriptional diversity of 25 Drosophila cell lines. Genome Res. 2011,21, 301–314. [CrossRef]
70.
Lee, H.; McManus, C.J.; Cho, D.-Y.; Eaton, M.; Renda, F.; Somma, M.P.; Cherbas, L.; May, G.; Powell, S.; Zhang, D.; et al. DNA
copy number evolution in Drosophila cell lines. Genome Biol. 2014,15, R70.
71.
Hammond Mp Fau-Laird, C.D.; Laird, C.D. Control of DNA replication and spatial distribution of defined DNA sequences in
salivary gland cells of Drosophila melanogaster.Chromosoma 1985,91, 279–286. [CrossRef] [PubMed]
72.
Spradling, A.; Orr-Weaver, T. Regulation of DNA replication during Drosophila development. Annu. Rev. Genet.
1987
,21, 373–403.
[CrossRef]
73. Goshima, G.; Wollman, R.; Goodwin, S.S.; Zhang, N.; Scholey, J.M.; Vale, R.D.; Stuurman, N. Genes required for mitotic spindle
assembly in Drosophila S2 cells. Science 2007,316, 417–421. [CrossRef] [PubMed]
74.
Lindström, M.S. Emerging functions of ribosomal proteins in gene-specific transcription and translation. Biochem. Biophys. Res.
Commun. 2009,379, 167–170. [CrossRef] [PubMed]
75.
Marsano, R.M.; Caizzi, R.; Moschetti, R.; Junakovic, N. Evidence for a functional interaction between the Bari1 transposable
element and the cytochrome P450 cyp12a4 gene in Drosophila melanogaster.Gene 2005,357, 122–128. [CrossRef]

Genes 2021,12, 1997 17 of 17
76.
Palazzo, A.; Caizzi, R.; Viggiano, L.; Marsano, R.M. Does the Promoter Constitute a Barrier in the Horizontal Transposon Transfer
Process? Insight from Bari Transposons. Genome Biol. Evol. 2017,9, 1637–1645. [PubMed]
77.
Palazzo, A.; Lorusso, P.; Miskey, C.; Walisko, O.; Gerbino, A.; Marobbio, C.M.T.; Ivics, Z.; Marsano, R.M. Transcriptionally
promiscuous “blurry” promoters in Tc1/mariner transposons allow transcription in distantly related genomes. Mob. DNA
2019
,
10, 13. [CrossRef] [PubMed]
78.
Suresh, S.; Ahn, H.W.; Joshi, K.; Dakshinamurthy, A.; Kananganat, A.; Garfinkel, D.J.; Farabaugh, P.J. Ribosomal protein and
biogenesis factors affect multiple steps during movement of the Saccharomyces cerevisiae Ty1 retrotransposon. Mob. DNA
2015
,
6, 22. [CrossRef]