Phosphorylation of the RNase III enzyme Drosha
at Serine300 or Serine302 is required for its
Xiaoli Tang, Yingjie Zhang, Lynne Tucker and Bharat Ramratnam*
Laboratory of Retrovirology, Division of Infectious Diseases, Department of Medicine, Warren Alpert Medical
School of Brown University, Providence, RI 02903, USA
Received January 29, 2010; Revised May 27, 2010; Accepted May 28, 2010
The RNaseIII enzyme Drosha plays a pivotal role in
microRNA (miRNA) biogenesis by cleaving primary
miRNA transcripts to generate precursor miRNA in
the nucleus. The RNA binding and enzymatic
domains of Drosha have been characterized and
are on its C-terminus. Its N-terminus harbors a
truncated Drosha constructs, we narrowed down
the segment responsible for nuclear translocation
to a domain between aa 270 and aa 390. We
further identified two phosphorylation sites at
Serine300 (S300) and Serine302 (S302) by mass
spectrometric analysis. Double mutations of S!A
at S300 and S302 completely disrupted nuclear
localization. Single mutation of S!A at S300 or
S302, however, had no effect on nuclear localization
indicating that phosphorylation at either site is suf-
ficient to locate Drosha to the nucleus. Furthermore,
mimicking phosphorylation status by mutating S!E
at S300 and/or S!D at S302 restored nuclear local-
ization. Our findings add a further layer of complex-
ity to the molecular anatomy of Drosha as it relates
to miRNA biogenesis.
MicroRNAs (miRNAs) are a class of endogenous
non-protein-coding small RNAs of ?22nt in length that
impact gene expression by sequence-specific interaction
with homologous mRNA (1). Presently, it is thought
that miRNAs most commonly repress gene expression
by base-pairing with the 30-untranslated region (UTR) of
their target mRNAs. However, recent work has also
identified miRNA that target coding sequences of genes
(2). Additionally, miRNA may also target promoter
regions and thereby act as pro-transcriptional elements,
as in the case of miRNA-373 and the gene encoding
E-cadherin (3). One specific miRNA may inhibit many
target genes and one specific gene may be regulated by
miRNA-125a and b, the genes of which are located on
different chromosomes, target the p53 protein as both
miRNAs harbor similar seed sequences that share similar-
ity to the p53 30UTR (4,5). MiRNAs play increasingly
recognized roles in several basic processes including cell
signal transduction, tumorigenesis, tumor invasion and
metastasis, stem cell renewal, immune function, apoptosis
and reaction to stress (6–11). The vast majority of miRNA
genes are thought to be under the control of RNAP II
with others being recently identified as substrates of
RNAP III (12,13). Irrespectively, miRNA genes are ini-
tially transcribed to yield a primary, long transcript that
undergoes successive processing in both the nucleus and
cytoplasm. Nuclear processing is mediated by the RNase
Critical Region Gene 8) microprocessor complex to
generate precursor miRNAs (pre-miRNAs) of ?70nt in
length. Pre-miRNAs are subsequently transported to the
cytoplasm by export 5-Ran-GTP where they are cleaved
by the RNase III enzyme Dicer to generate mature
Investigation into the molecular mechanisms of miRNA
biogenesis at the transcriptional and translational levels
has been intensively pursued (19–22). Drosha plays a
central role in miRNA biogenesis and recent work
suggests that its expression level directly influences
clinical outcomes in malignant disease thus underlying
the importance of better understanding mechanisms that
impact Drosha expression and function (23). Along these
lines, Drosha has been found to interact with other host
proteins including DGCR8. Drosha and DGCR8 regulate
each other post-transcriptionally. The Drosha–DGCR8
complex destabilizes DGCR8 mRNA by cleaving its
hairpin structure. DGCR8, in turn, stabilizes Drosha via
*To whom correspondence should be addressed. Tel: +1 401 444 5219; Fax: +1 401 444 2939; Email: email@example.com
Nucleic Acids Research, 2010, Vol. 38, No. 19Published online 16 June 2010
? The Author(s) 2010. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
protein–protein interaction (22). In an elegant experiment,
deletion of Drosha N-terminal 390 amino acids had no
effect on its binding with DGCR8 and its ability to
process pri-miRNA in vitro (21). While these results sug-
gested that the N-terminal region is dispensable vis-a-vis
the protein’s catalytic activity, they also raised an interest-
ing question: what role, if any, does N-terminal sequence
play in Drosha function in vivo?
Here, we examine the subcellular localization of
wild-type Drosha and N-terminally deleted Drosha. We
find that wild-type Drosha locates to the nucleus, as
expected, but Drosha with N-terminal 390 amino acids
deleted is located exclusively in the cytoplasm. These
results were in agreement with previous work which
found that the N-terminus of Drosha is not dispensable
but rather essential for its nuclear localization and
ultimate miRNA processing function (24). We further
narrowed down the domain for nuclear localization to
the N-terminal region between aa 270 and aa 390. We
show by microscopy and functional assays that phosphor-
ylation at Serine300 or Serine302 is absolutely required for
the nuclear localization and subsequent miRNA process-
ing activities of Drosha.
MATERIALS AND METHODS
Plasmids and primers
Thomas Tuschl and deposited at Addgene (Addgene
plasmid 10828; 25). Wild-type and mutated Drosha were
subcloned into pEGFP-C1 vector (GenBank Accession
#:U55763) using restriction endonucleases HindIII and
cat.#R0142L, respectively). Point mutants were generated
with site-directed mutagenesis techniques as previously
described (26). The primers used for PCR were ordered
from Integrated DNA Technologies. The primer se-
psiCHECK2-miRNA-143 plasmid was created by insert-
ing antisense target sequences of miRNA-143 ds-oligos
AGCGGCCGCAAAAGGAAAA and antisense: TTTTC
CCTCGAGCGG) into psiCHECK2 vector (Promega)
using NotI and XhoI (New England Biolabs) restriction
endonuclease sites. All the cDNA constructs were verified
Biotechnology Resource Laboratory.
was originally created by
Yale KECK Foundation
Antibodies and reagents
antibody (sc-33778), Anti-IkB-a (C-21) rabbit polyclonal
antibody (sc-371), anti-Lamin A (H-102) rabbit poly-
clonal antibody (sc-20680), anti-GFP (B-2) mouse mono-
clonal antibody (sc-9996) and Protein G Plus-Agarose
Biotechnology. Pfu DNA Polymerase (cat.#600136) and
Stratagene. Lipofectamine Reagent (cat.#18324-020) and
III Drosha(H-300) rabbitpolyclonal
purchased from Invitrogen. Alexa Fluor 680 goat anti-
rabbit IgG (H&L) (cat.#A21076) and Hoechst 33342
(cat#H3570) were purchased from Invitrogen Molecular
Probes. IRDye800-conjugated Affinity Purified Anti-
Mouse IgG (H&L) (cat.#610-132-121) was purchased
from Rockland Immunochemicals.
(cat.#BP-190) were purchased from Boston Bioproducts.
LB Agar Kanamycin-50 Plates (cat.#L0643) and EDTA
(cat.#E7889) were purchased from Sigma. The 10?Tris-
Buffered Saline (cat.#170-6435)
solution (cat.#161-0781) were purchased from Bio-Rad
Inhibitor Cocktail Tablets (cat.#11 836 170 001) was
produced by Roche Diagnostics GmbH. QIAprep Spin
Miniprep Kit (cat.#27106), QIAquick PCR Purification
Kit (cat.#28106) and MinElute Gel Extraction Kit
(cat.#28604) were purchased from QIAGEN. Coomassie
Brilliant Blue R-250 Dye (cat.#20278) was purchased from
and 10% Tween20
Cell culture and transfection
HEK293T and Huh-7 cells were cultured in Dulbecco’s
modified Eagle’s medium (Invitrogen, Carlsbad, CA,
USA) with 10% fetal bovine serum and 2mM L-glutam-
ine. HeLa cells were grown in Eagle’s Minimum Essential
Medium supplemented with 10% fetal bovine serum,
2mM L-glutamine and non-essential amino acids. Cells
were trypsinized and reseeded in culture plates 1day
before transfection. HEK293T transfection was per-
formed with Lipofectamine when cell confluency was
?60%. Huh-7 and HeLa cells were transfected with
Lipofectamine 2000 when cell confluency was ?80%.
Cytoplasmic and nuclear fractionation
Cytoplasmic and nuclear fractionation was performed
using EZ Nuclei Isolation Kit (Sigma, cat.#NUC-101) ac-
cording to the manufacturer’s instructions. Briefly, cells
were harvested and washed once with cold PBS. The
cells were then suspended in EZ Nuclei Isolation buffer
and rotated at 4?C for 5min. After centrifugation at 4?C
for 5min, supernatant was collected containing the cyto-
plasmic fraction. Cell lysis and centrifugation were
repeated three times. The final pellets were collected as
the nuclear fraction and lysed in modified RIPA buffer
(50mM Tris–HCl pH 7.4, 150mM NaCl, 2mM EDTA,
1% NP-40, 1% Triton X-100).
Cell lysates (100mg protein each) were separated by 10%
SDS–PAGE electrophoresis and electroblotted to nitrocel-
lulose membrane (Bio-Rad, cat.#162-0115). Blotted mem-
antibodies rotating at 4?C overnight. The membranes
were washed three times in TBST buffer and probed
with secondary antibody (Alexa Fluor 680 goat anti-
rabbit IgG or IRDye800-conjugated Affinity Purified
Anti-Mouse IgG, respectively) at room temperature
for 1h. Membranes were then washed three times in
TBST buffer and direct infrared fluorescence detection
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