Regulatory RNAs derived from transfer RNA?
ABSTRACT Four recent studies suggest that cleavages of transfer RNAs generate products with microRNA-like features, with some evidence of function. If their regulatory functions were to be confirmed, these newly revealed RNAs would add to the expanding repertoire of small noncoding RNAs and would also provide new perspectives on the coevolution of transfer RNA and messenger RNA.
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ABSTRACT: BackgroundtRFs, 14¿32 nt long single-stranded RNA derived from mature or precursor tRNAs, are a recently discovered class of small RNA that have been found to be present in diverse organisms at read counts comparable to miRNAs. Currently there is a debate about their biogenesis and function.ResultsThis is the first meta-analysis of tRFs. Analysis of over 50 short RNA libraries has revealed that tRFs are precisely generated fragments present in all domains of life (bacteria to humans), and are not produced by the miRNA biogenesis pathway. Human PAR-CLIP data shows a striking preference for tRF-5s and tRF-3s to associate with AGO1, 3, and 4 rather than AGO2, and analysis of positional T to C mutational frequency indicates these tRFs associate with Argonautes in a manner similar to miRNAs. The reverse complements of canonical seed positions in these sequences match cross-link centered regions, suggesting these tRF-5s and tRF-3s interact with RNAs in the cell. Consistent with these results, human AGO1 CLASH data contains thousands of tRF-5 and tRF-3 reads chimeric with mRNAs.ConclusionstRFs are an abundant class of small RNA present in all domains of life whose biogenesis is distinct from miRNAs. In human HEK293 cells tRFs associate with Argonautes 1, 3 and 4 and not Argonaute 2 which is the main effector protein of miRNA function, but otherwise have very similar properties to miRNAs, indicating tRFs may play a major role in RNA silencing.BMC Biology 10/2014; 12(1):78. · 7.43 Impact Factor
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ABSTRACT: A set of small RNAs known as rasRNAs (repeat-associated small RNAs) have been related to the down-regulation of Transposable Elements (TEs) to safeguard genome integrity. Two key members of the rasRNAs group are piRNAs and endo-siRNAs. We have performed a comparative analysis of piRNAs and endo-siRNAs present in mouse oocytes, spermatozoa and zygotes, identified by deep sequencing and bioinformatic analysis. The detection of piRNAs and endo-siRNAs in spermatozoa and revealed also in zygotes, hints to their potential delivery to oocytes during fertilization. However, a comparative assessment of the three cell types indicates that both piRNAs and endo-siRNAs are mainly maternally inherited. Finally, we have assessed the role of the different rasRNA molecules in connection with amplification processes by way of the “ping-pong cycle”. Our results suggest that the ping-pong cycle can act on other rasRNAs, such as tRNA- and rRNA-derived fragments, thus not only being restricted to TEs during gametogenesis.Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 01/2014; · 5.44 Impact Factor
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ABSTRACT: High-throughput sequencing reveals an abundance of microRNA-sized fragments derived from larger non-coding RNAs. Roles for these small RNAs in gene silencing are suggested by their co-precipitation with Argonaute, the microRNA effector protein, though the extent to which they suppress gene expression endogenously remains unclear. To address this, we used luciferase reporters to determine the endogenous functionality of small RNAs from a diverse range of sources. We demonstrate small RNAs derived from snoRNAs have the capacity to act in a microRNA-like manner, though we note the vast majority of these are bound to Argonaute at levels below that required for detectable silencing activity. We show Argonaute exhibits a high degree of selectivity for the small RNAs with which it interacts and note that measuring Argonaute-associated levels is a better indicator of function than measuring total expression. Although binding to Argonaute at sufficient levels is necessary for demonstrating microRNA functionality in our reporter assay, this alone is not enough as some small RNAs derived from other non-coding RNAs (tRNAs, rRNAs, Y-RNAs) are associated with Argonaute at very high levels yet do not serve microRNA-like roles. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.Nucleic Acids Research 12/2014; · 8.81 Impact Factor
Regulatory RNAs derived from transfer RNA?
1Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605 USA
2Program in Cell and Developmental Dynamics, University of Massachusetts Medical School, Worcester, Massachusetts 01605 USA
Four recent studies suggest that cleavages of transfer RNAs generate products with microRNA-like features, with some evidence of
function. If their regulatory functions were to be confirmed, these newly revealed RNAs would add to the expanding repertoire
of small noncoding RNAs and would also provide new perspectives on the coevolution of transfer RNA and messenger RNA.
Keywords: Argonautes; deep sequencing; microRNAs; transfer RNA
DEEP SEQUENCING REVEALS TRANSFER
RNA-DERIVED REGULATORY RNAS
The discovery of microRNAs was a milestone in the modern
era of biology (Lee et al. 1993; Wightman et al. 1993). The
many hundreds of microRNAs in an organism are processed
RNAs, or from genes that have evolved to produce only
microRNAs. Related pathways produce endogenous small
interfering RNAs and the germline-expressed piRNAs
(Ghildiyal and Zamore 2009). Now, four nearly contem-
poraneous papers have revealed another class of small non-
coding RNAs with microRNA features. They are derived
from a pioneer entry in the RNA discovery chain: transfer
RNA (Hoagland 2004).
There had been previous reports of transfer RNA-derived
fragments (Lee and Collins 2005; Calabrese et al. 2007;
Babiarz et al. 2008; Kawaji et al. 2008). But, the new work
reviewed here took this to a more refined analytical depth
function of transfer RNA-derived microRNA-like RNAs.
First in the current wave of studies was an investigation of
small RNAs in HIV-infected cells (Yeung et al. 2009). In
addition to detecting several small noncoding RNAs that
were known from previous work to be processed from the
viral RNA itself, this study revealed the presence in HIV-
infected cells of a small RNA corresponding to nucleotides
(PBS) in the genomic HIV, where it serves as the primer for
reverse transcription. Additional experiments demonstrated
that the cellular prevalence of this 20-nucleotide (nt) RNA
derived small RNA was bound to a canonical Argonaute
protein, Ago 2; that it could silence a luciferase reporter en-
this RNA to the HIV RNA PBS is a substrate for Dicer
cleavage in vitro.
Only 2 mo later, Lee et al. (2009) reported a deep-
sequencing analysis of total small RNAs from two human
prostatic carcinoma cell lines. They got >600,000 reads that
included 635 out of the 695 microRNAs in the Sanger
database. Among the remainder they found 17 RNAs, 18–
22 nt in length, that aligned with transfer RNA sequences.
Five were derived from the 59 ends of mature tRNAs, eight
were derived from the 39 ends of mature tRNAs, and four
were derived from the 39 trailer regions of pre-tRNAs. The
cloning coverage indicated that these tRNA-related small
RNAs are more abundant in these cells than the majority
of microRNAs, and are within an order of magnitude as
abundant as the most prevalent microRNAs. Spirited by
these findings, Lee et al. (2009) dove back into the pot of
all the other noncanonical microRNAs in their library (i.e.,
ones not in the Sanger database) and turned up another 621
that correspond to tRNA sequences (this large number
smaller number of tRNA species).
of their RNAs, tRF-1001, which corresponds to the 39 trailer
of pre-tRNASerTGA. They found that it displayed an elevated
and tissues and that, among the cell lines, its expression
was correlated with proliferation rate. They succeeded in
Reprint requests to: Thoru Pederson, Department of Biochemistry and
Molecular Pharmacology, University of Massachusetts Medical School,
Worcester, MA 01605 USA; e-mail: firstname.lastname@example.org;
fax: (508) 856-8668.
Article published online ahead of print. Article and publication date are
RNA (2010), 16:1865–1869. Published by Cold Spring Harbor Laboratory Press. Copyright ? 2010 RNA Society.
knocking down tRF-1001 with siRNA (this itself was in-
and found that this slowed cell proliferation. This effect was
blocked by the cointroduction of a 29-O-methyl version of
the production of tRF-1001 involves ELAC2, a homolog of
the pre-tRNA 39-processing enzyme RNase Z, which is com-
patible with the fact that tRF-1001 is derived from a tRNA
(2009) reported a deep-sequencing study in HeLa cells that
also revealed numerous RNAs corresponding to transfer
RNA fragments. Most of their reads corresponded to the
study and showed that it is cleaved by Dicer both in vivo and
in vitro, although one might have hoped that they would
have examined this with respect to more than a single tRNA.
They went on to find that this tRNAGln-derived RNA was
more sensitive to knockdown of Dicer than were bona fide
microRNAs, although the possibility was not examined that
this hypersensitivity might reflect a general destabilization
of tRNA due to a slowing of growth resulting from Dicer
knockdown. They further found that although their tRNA-
derived RNAs were bound to Ago 1 and Ago 2 to some de-
gree, this complexing was much less stoichiometric than for
canonical microRNAs. Moreover, Cole et al. (2009) found
that these tRNA-derived RNAs carried blocked 29-OH
termini. In all of these respects, these tRNA-derived RNAs
differ from typical mammalian microRNAs. Thus, although
these findings, taken together, were indeed provocative, the
picture remained rather unsettled.
Subsequently (4 mo later), Haussecker et al. (2010)
reported a study of small noncoding RNAs in the adenovi-
rus-transformed human embryonic kidney 293 cell line,
which again revealed transfer RNA-derived species. Owing
a combined decapping and RNA ligation strategy. For our
purposes, the decapping aspect is irrelevant (as it proved to
of T4 RNA ligase to covalently circularize RNAs bearing
a 59 phosphate and a 39 hydroxyl (and to also conjoin two or
more such molecules, which is beyond the scope of our
discussion here). This assay is based on the resulting elec-
trophoretic gel shift (or disappearance) of certain RNA spe-
cies. Using this approach they observed two populations of
tRNA-derived RNAs. One corresponded to the 39 ends of
mature tRNAs. Their presence was found to be be Dicer
dependent. Haussecker et al. (2010) termed these ‘‘Type I.’’
The second population of tRNA-derived RNAs corre-
sponded to the 39 ends of pre-tRNAs, and this group was
termed ‘‘Type II.’’ Their production was Dicer independent
and presumably involves the action of RNase Z in the
standard pre-tRNA 39 processing pathway.
The type I RNAs were found to be complexed with
Argonautes 1–4, but not Mov10. In contrast, the type II
RNAs were found to be complexed with Argonautes 3 and 4
all to Mov 10. Haussecker et al. (2010) further reported that
of Type II tRNA-derived RNAs increased. But, as silencing
experiments were undertaken with standard luciferase re-
porters, things began to get even more interesting.
When a dual Renilla-Firefly luciferase construct bearing
a target for one of the Type I RNAs, ‘‘cand14,’’ was trans-
fected into cells expressing this RNA, the addition of an
antisense oligo to cand14 resulted in a modest (30%–40%)
elevation of reporter expression, consistent with a role of
this RNA in silencing. (The investigators speculated that the
limited silencing observed might reflect the preferential
association of cand14 with Ago 3 and Ago 4, relative to
Ago 1 or Ago 2, as well as other possible factors.) In contrast
to these results with cand14, when a comparable experi-
ment was performed with cells expressing a Type II RNA,
‘‘cand45,’’ and a reporter containing a cand45 target, the
addition of an antisense oligo to cand45 did not result in
an elevation of reporter expression, indicating that, unlike
cand14, cand45 does not play a role in silencing. The in-
vestigators considered various possibilities for this contrast-
ing finding and decided, as one potential approach, to
overexpress cand45, reasoning that perhaps its prevalence
in these cells simply was inadequate relative to the reporter
target levels being expressed. However, this still did not lead
the addition of the oligo antisense to cand45 actually in-
creasedsilencing, aphenomenonthey termed sense-induced
They further found that SITS involved all three cand45
target sites in the reporter and that it was not dependent
on the chemistry of the antisense oligo used—a completely
substituted ribose-29-O-methyl oligo was as effective as
the initial cand45 oligo they had used, which contained a
trio of unmodified deoxy, ribose-29-O-methyl and locked
nucleic acid nucleotides. Indeed, the fully substituted
29-O-methyl antisense oligo was observed to induce re-
porter silencing even when cand45 was not overexpressed.
Finally, Haussecker et al. (2010) also observed SITS in a
mouse embryonic cell line, indicating that this phenomenon
is not a quirk of human 293 cells. Though presently unex-
plained as to mechanism, these provocative findings point
to a distinction between the messenger RNA action of
certainof these tRNA-derived RNAs versus that of canonical
microRNAs, and could possibly be the most important
finding Haussecker et al. (2010) made in their study.
A deeper look at deep sequencing
Current RNA cloning and deep sequencing methods are so
sensitive that one expects virtually every RNA fragment
RNA, Vol. 16, No. 10
present in a cell at a few copies (maybe even one) to be
captured (unless either end is blocked from taking the
adaptor linkers and cannot be enzymatically converted into
an unblocked end). So, obviously, the first question is
whether these tRNA-related small RNAs are simply a
The results argue against this hypothesis. As mentioned, Lee
et al. (2009) found that five of the RNAs aligned with the
59 ends of various tRNAs, eight corresponded to tRNA
39 ends, and four mapped to the 39 trailers of pre-tRNAs.
loop regions of tRNAs. The degree of nonrandomness was
even greater for the RNAs reported by Cole et al. (2009),
which predominantly mapped to the 59 ends of mature
tRNAs and extending 39-ward to just beyond the D-loop.
Moreover, these investigators noted a noncorrelation (in-
deed, an anticorrelation) between the prevalence of reads in
the deep sequencing and the known abundance of various
tRNAs in HeLa cells, and a comparable noncorrelation with
the anticodon prevalence of isoaccepting tRNAs. Moreover,
do not correspond to the prevalence of these amino acids in
HeLa cell proteins. In addition, the data of Haussecker et al.
(2010) also point to these tRNA-derived RNAs as being
nonrandom cleavage products.
Bona fide microRNAs?
One of course next asks: Are they really microRNAs? We can
first ask: What is the definition of a microRNA? If the
definition is that of an RNA derived from a pol II transcript
by Drosha processing in the nucleus, export of a pre-micro-
RNA and Dicer action, then these tRNA-derived RNAs are
disqualified from the outset since they are pol III transcripts.
But the evidence for processing of at least some of them by
Dicer seems provocative, so by that criterion those may
the evidence is a bit wobbly among the studies, but there is
nevertheless a sense of connectivity. The experimental re-
direction of one of these RNAs into Ago 2 complexes (Cole
et al. 2009) seems provocative as to both function and
interactivity with the canonical microRNA pathway. Mean-
while, the finding of blocked 39 termini on these RNAs,
presumed (but not demonstrated to be) ribose-29-O-methyl
modification has so far not been found in mammalian
microRNAs. This suggests that these tRNA-derived RNAs
are perhaps siblings, but not identical twins, of bona fide
The starkest deficiency in all of these four studies was the
lack of mRNA target identification for these tRNA-derived
RNAs. There can be no meaningful progress until this is
done. Target identification is a challenging bioinformatics
endeavor but should be no more so for these tRNA-derived
RNAs than for the extant microRNA databases that have
been so analyzed to date.
A curious twist is that, as chance would have it, the very
‘‘Type II’’ tRNA-derived RNA chosen for scrutiny by
Haussecker et al. (2010) (cand45) is none other than tRF-
1001, as investigated by Lee et al. (2009). To the extent that
comparable experiments were performed in the two studies,
delve far enough to discover SITS). That said, in today’s very
fast moving small regulatory RNA field we might not want
to jump too quickly into holding up these four manuscripts
as necessarily constituting an international tribunal of con-
sensus. The evidence that these transfer RNA-derived RNAs
are actually microRNAs presently hinges on the criteria of
Argonaute association and thesilencing results, the latterless
new Argonautes and related proteins awaiting discovery.)
And why were these tRNA-derived RNAs not seen in other
preceding year or so? Perhaps in previous studies the in-
vestigators decided to simply not pursue reads that were not
fully consider other possibilities in their Discussion.
On this front (of interest to both the longstanding RNA
processing community as well as the more recently expand-
ing small regulatory RNA field), we presently have more
questions than answers. How do the cleavages predicted to
produce these RNAs navigate around the canonical process-
ing enzymes that would also be engaging these molecules,
that is, RNase P and RNase Z at the 59 and 39 termini of pre-
tRNAs, respectively, and subsequently, the aminoacyl-tRNA
synthetases? Where in the cell are these tRNA-derived RNAs
produced? 59 and 39 processing of tRNA takes place in the
nucleus (Hopper et al. 2010), but where do the cleavages
needed to produce these observed tRNA-derived regulatory
RNAs occur? Dicer cleavage of pre-microRNAs (70-nt long,
hauntingly similar to the length of tRNAs, but with one loop
versus four in the latter) is thought to be cytoplasmic, but to
the extent that some of these newly discovered RNAs are the
39 trailers of pre-tRNAs, the nucleus is implicated as the site
derived small RNAs might compete with other processes in
which full-length tRNAs have been implicated, for example,
is also of interest to consider how these new findings might
bear on cases in which engineered tRNAs are expressed in
other functions, for example, as chimeras with ribozymes
(Geslain et al. 2009). Would such modified tRNAs also pro-
cerning the RNA processing horizon of these new findings.
Transfer RNAs and microRNAs
A surprising recent finding in the tRNA biosynthesis field
and Shaheen 2008). In light of the new findings reviewed
here, is it possible that these returning RNAs get cleaved in
the nucleus? If so, would they be exported as microRNA-like
RNAs or might they perhaps perform nuclear functions?
Within the nucleus, nucleoli were long ago discovered to
harbor tRNAs (Sirlin 1972). Certain microRNAs have re-
these are ones that are produced via Drosha processing of
typical pri-microRNA precursors. The classical evidence for
findings in support of this (Bertrand et al. 1998; Thompson
et al. 2003) raise the question of whether tRNAs are cleaved
into microRNA-like RNAs in the nucleolus.
Another question is how the coding functions of a tRNA
are balanced with its role as a precursor of small regulatory
RNAs. For those of the latter that are derived from the 59 or
39 ends of pre-tRNAs, clearly their precursors do not ever
function in coding. But for those that are derived from
mature tRNAs, does this occur only after they have been
aminoacylated in one or more rounds of translation? If so,
what then determines their fate as precursors of small
regulatory RNAs? Alternatively, if there are special subsets
of tRNAs that are never subject to aminoacylation, but are
sent directly into the small regulatory RNA production
pathway (seemingly the less likely of the two possibilities),
of interest to note that in S. pombe there is a mechanism that
siRNA biogenesis pathway (Buhler et al. 2008). It is conceiv-
able that such a mechanism operates in higher eukaryotes as
well as under some conditions and perhaps happens to be
relaxed or absent in the cultured cell lines in which these
recent deep-sequencing studies were carried out. Alterna-
tively, the S. pombe mechanism may have been selected
against during the evolution of higher eukaryotes, due to a
need to maximize all possible sources of small regulatory
RNAs to serve the greater complexity of mRNA targets and
build the necessarily more extensive RNA-mediated trans-
broad phyletic distribution of tRNA-derived small regula-
tory RNAs will begin to come into view.
Are there other ‘‘unconventional’’ pre-microRNAs?
If tRNAs produce regulatory RNAs,might othersmall RNAs
do so aswell? Recent studies have pointed to microRNA-like
RNAs derived from certain of the small nucleolar RNAs
(Ender et al. 2008; Kawaji et al. 2008; Scott et al. 2009; Taft
are components of a ribonucleoprotein machine involved
in multidrug resistance, have recently been demonstrated
to produce a group of z23-nt RNAs (Persson et al. 2009).
Although this processing step is Dicer independent, these
vault RNA-derived RNAs include one that is Argonaute
bound and mediates mRNA cleavage and, hence, is a micro-
RNA by those criteria. One wonders how many other small
RNAs beyond tRNAs, snoRNAs, and vault RNAs may be cut
miRNAs and subsequent Dicer-mediated production of
mature microRNAs. Perhaps pol III transcripts other than
tRNAs and vault RNA are cleaved to produce regulatory
RNAs? The candidates would include the small Ro RNAs
(Hendrick et al. 1981), the signal recognition particle RNA
(Walter and Blobel 1982), U6 spliceosomal RNA (Kunkel
et al. 1986), the RNA components of RNase P and RNase
MRP (Chang and Clayton 1989; Baer et al. 1990), as well as
the large number of pol III transcripts produced by retro-
transposons. These possibilities are just guesses at present,
answers soon enough. Meanwhile, and notwithstanding the
diversity of the RNA processing pipeline that may be
ultimately revealed, the recent evidence for tRNA-derived
regulatory RNAs reviewed here alerts us to possibilities not
previously pondered as we move along the ‘‘new RNA
world,’’ a seemingly endless frontier in our time.
Transfer RNA evolution re-examined
aminoacylation specificity (McClain 1993; Schimmel and
Ribas de Pouplana 1995). These dual, cooperating motifs
that determine aminoacylation specificity consist of the
very 59 and 39 regions of tRNAs that are so emphatically
represented in the deep-sequencing studies reviewed here.
This surely cannot be ignored. The fact that these tRNA-
derived regulatory RNAs tend less often to be derived from
the central regions might reflect the fact that the anticodons
are configured optimally for base pairing, and would thus
likely bind codons all throughout an mRNA. This suggests
selected for in the evolution of these newly described RNAs
and that the anticodon regions simply had no selective
advantage for these mRNA regulatory functions (as opposed
to coding) on which selection was operating. One thus sus-
pects that there was nested coevolution of four entities—
the anticodons, the aminoacylation code (residing in the 59
end and the 39 acceptor stem), the aminoacyl-tRNA synthe-
tases, and the domains producing small regulatory RNAs.
The alternative possibility, viz., that the latter function arose
later as what evolutionary nomenclature calls an exaptation,
seems less likely. What remains then is the question of what
selective pressures were brought upon the other regions
of tRNAs and whether or not they are more than mere
evolutionary ‘‘spandrels.’’ In this context it is noteworthy
that some of the RNAs described by Lee et al. (2009) that
RNA, Vol. 16, No. 10
come from the 59 ends of mature tRNAs included the D
Finally, the 59 and 39 elements of the investigated tRNA
minihelices that have given rise to the aminoacylation
specifity code are, of course, base paired in their parental
their concentrations in the cell be sufficiently high to lead to
base pairing? If so, then these duplexes of tRNA-derived
RNA-dependent RNA polymerases and the production of
secondary RNAs as in RNA interference. Are such possibil-
ities among the new things under the sun awaiting discovery
in the regulatory RNA field?
I am grateful to Paul Schimmel (The Scripps Research Institute)
for encouragement on an early draft and for reinforcing the fact
that these RNAs are mostly derived from tRNA regions that
constitute the aminoacylation specificity code. I thank Victor
Ambros (University of Massachusetts Medical School), Andrew
Fire (Stanford University School of Medicine), and Phillip Sharp
(M.I.T.) for critically commenting on an initial draft.
The discoverers of transfer RNA, Mahlon Hoagland (1920–2009)
and Paul Zamecnik (1912–2009), were close colleagues of the
investigator, who knows how keenly interested they would have
been in these new findings. They almost lived to see them.
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