the multifunctional rna-binding protein hnrnp a1 is required for processing of mir-18a
TRANSCRIPT
The multifunctional RNA-binding protein hnRNP A1is required for processing of miR-18aSonia Guil & Javier F Caceres
hnRNP A1 is an RNA-binding protein involved in various aspects of RNA processing. Use of an in vivo cross-linking andimmunoprecipitation protocol to find hnRNP A1 RNA targets resulted in the identification of a microRNA (miRNA) precursor,pre-miR-18a. This microRNA is expressed as part of a cluster of intronic RNAs, including miR-17, miR-18a, miR-19a, miR-20a,miR-19b-1 and miR-92, and potentially acts as an oncogene. Here we show that hnRNP A1 binds specifically to the primary RNAsequence pri-miR-18a before Drosha processing. HeLa cells depleted of hnRNP A1 have reduced in vitro processing activity withpri-miR-18a and also show reduced abundances of endogenous pre-miR-18a. Furthermore, we show that hnRNP A1 is requiredfor miR-18a–mediated repression of a target reporter in vivo. These results underscore a previously uncharacterized role forgeneral RNA-binding proteins as auxiliary factors that facilitate the processing of specific miRNAs.
A key feature of post-transcriptional control of gene expression isthe association of newly transcribed messenger RNAs with a largevariety of RNA-binding proteins, forming messenger ribonucleo-protein complexes (mRNPs). These mRNP particles influence notonly pre-mRNA splicing but also RNA transport and cytoplasmicevents1. The RNA-binding proteins involved in these processes arecollectively known as hnRNP proteins. In humans, this family ofproteins consists of at least 20 different polypeptides. Among them,hnRNP A1, a nucleo-cytoplasmic shuttling protein that is involved inmany aspects of mRNA metabolism, has been extensively studied. Ithas been shown that hnRNP A1 and other members of the hnRNP A/Bfamily of proteins function in alternative splicing regulation as anantagonist of the SR family of proteins, both in vitro and in vivo2–5.It has recently been proposed that hnRNP A/B and hnRNP F/Hproteins may also function in generic splicing by modulating theconformation of mammalian pre-mRNAs6. hnRNP A1 shuttles con-tinuously between nucleus and cytoplasm7 and has been shown tofunction in various post-splicing activities such as mRNA export8,internal ribosome entry site (IRES)-mediated translation9 andmRNA stability10.
Despite its extensive characterization, only a relatively small numberof cellular and viral genes regulated by hnRNP A1 have beenidentified11–18, and only a small number of high-affinity bindingsites have been obtained by SELEX19. We used an in vivo cross-linkingand immunoprecipitation protocol (CLIP) to search for endogenousRNA targets of hnRNP A1. Here we report that hnRNP A1 bindsspecifically to human pri-miR-18a (the stem-loop precursor ofmiR-18a in the context of the primary RNA) and facilitates itsDrosha-mediated processing. Thus, hnRNP A1 acts as an auxiliaryfactor for the processing of a specific miRNA substrate.
RESULTSHnRNP A1 binds specifically to pri-miR-18aTo identify physiologically relevant RNA targets of hnRNP A1, we usedCLIP experiments20,21 to identify RNA targets directly bound tohnRNP A1. In this method, irreversible in vivo cross-linking isfollowed by highly stringent immunoprecipitation conditions, sothat only those RNAs directly bound to the protein of interest areselected. We unexpectedly found one miRNA, miR-18a, among thehnRNP A1 RNA targets. It should be noted that this was the onlymiRNA identified among 200 hnRNP A1 targets obtained by the CLIPprotocol (S.G. and J.F.C., unpublished data). miRNAs are smallnoncoding RNA gene products approximately 22 nucleotides in lengththat negatively regulate the expression of complementary mRNAs22.They are processed in the nucleus by an RNase III–type enzyme,termed Drosha, resulting in the production of stem-loop intermedi-ates, known as pre-miRNAs23,24. Subsequent maturation steps includeexport of pre-miRNAs25 and their processing in the cytoplasm by theenzyme Dicer, rendering the mature miRNAs26,27. More than half ofall known mammalian miRNAs are encoded in introns of eitherprotein-coding or noncoding transcription units28.
According to CLIP analysis, hnRNP A1 binds the stem-loop pre-cursor of miR-18a, which we refer to as pre-miR-18a. The mir-18agene is a component of the mir-17 cluster of an intronic miRNApolycistron. This cluster encodes six precursor miRNAs on chromo-some 13 (mir-17, mir-18a, mir-19a, mir-20a, mir-19b-1 and mir-92,abbreviated mir-17–92) and is closely related to two other clusters ofmiRNAs located in different genomic loci (mir-106, mir-18b, mir-20b,mir-19b-2 and mir-92-2, or mir-106–92-2, on chromosome X; andmir-106b, mir-93 and mir-25, or mir-106b–25, on chromosome 7)29.The mir-17 cluster is located on a region of DNA that is amplified
Received 24 October 2006; accepted 9 April 2007; published online 10 June 2007; doi:10.1038/nsmb1250
Medical Research Council Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, Scotland, UK. Correspondence should be addressed to J.F.C.([email protected]).
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in human B-cell lymphomas30 and has also been proposed to beinvolved in neovascularization in colon cancers31. The host gene,MIRH1, also called C13orf25, is unlikely to encode a protein, as itspredicted open reading frames (ORFs) encode only short peptides,which are not conserved in closely related species (Fig. 1a). Thus, themain consequence of DNA amplification would be an increase in theabundance of mature miRNA species from the mir17 cluster. Indeed,overexpression of the mir-17–mir-18a–mir-19b-1 cluster acceleratesMyc-induced tumor development in a mouse B-cell lymphomamodel32. Processing of the mir-18a precursor stem-loop, whichincludes the sequence bound by hnRNP A1 in CLIP assays, producestwo mature miRNAs: miR-18a and miR-18a* (Fig. 1b; the RNA foundin the CLIP assay, called the CLIP tag, is shown in bold)33.
To identify at which step hnRNP A1 binds this miRNA, weperformed immunoprecipitations with a specific antibody (4B10)and analyzed the bound RNAs by reverse-transcription PCR(RT-PCR) with specific primers that amplified the cluster mir-17–92or the control cluster mir-23–24-2 (Fig. 2a, see diagram). We couldspecifically amplify the mir-17–92 product from the immunoprecipi-tate, but not the mir-23–24-2 fragment (Fig. 2a, compare lanes 1 and5). This confirms the specificity of the interaction with the formercluster and implies that hnRNP A1 binds the primary sequence of themir-17 cluster before the cleavage by Drosha and the production ofindividual pre-miRNAs.
We next used RNA affinity purification as another means ofassessing which portion of the cluster is bound by hnRNP A1. An
in vitro–transcribed RNA termed pri17–19a, corresponding to theprimary mir-18a sequence (pri-miR-18a) and surrounding pri-miR-17and pri-miR-19a sequences, was covalently coupled to activatedagarose beads and incubated in the presence of HeLa nuclear extracts.As a control, primary sequence from the mir-23–24-2 cluster (pri23–24.2) was given the same treatment. Analysis of the retained proteinsby western blotting showed that hnRNP A1 was efficiently pulleddown with the pri17–19a sequence, whereas there was no binding tothe control pri23–24.2 RNA (Fig. 2b, lanes 3 and 4). By contrast, otherRNA-binding proteins, such as the SR protein SF2/ASF and thehnRNP K/J proteins, did not interact with either pri-miRNAsequence. Thus, hnRNP A1 is able to bind the 5¢ end of the miR-17cluster. Moreover, we found that in vitro, hnRNP A1 binds to thestem-loop pre-miR-18a much more efficiently than to pre-miR-19a(Fig. 2c, compare lanes 3 and 4). Together, these results suggestthat hnRNP A1 binds specifically to the miR-18a sequence beforeDrosha cleavage.
hnRNP A1 facilitates in vitro processing of pri-miR-18aWe hypothesized that hnRNP A1 could be acting at the level ofalternative splicing of the intron of the host gene; or, alternatively, itcould be directly involved in facilitating Drosha-mediated processing.To address this, we used the same primary miRNA sequences as inFigure 2b to set up in vitro processing assays independent of theintronic context of the host gene34. The sequence pri17–19a wasreadily processed in vitro in the presence of HeLa extracts (Fig. 3a,lane 3), rendering a product of B65 nucleotides (nt) that correspondsto pre-miR-18a (as revealed by northern blot analysis, SupplementaryFig. 1 online). This is the only pre-miRNA that is generated from theprimary substrate in appreciable amounts, presumably because theshort length of the flanking single-stranded RNA sequences surround-ing pre-miR-17 and pre-miR-19a do not allow efficient processing ofthese other two miRNAs35. In support of this, the higher–molecularweight bands (of about 200 and 150 nt) observed are similar in size tothe intermediates expected if only the central pre-miR-18a is cropped(193 and 147 nt, respectively; the much larger number of radiolabelednucleotides in the sequence would account for the stronger signal inthe B200-nt band). However, when the same substrate was incubatedwith extracts made from HeLa cells depleted of hnRNP A1 by RNAinterference (RNAi), the production of pre-miR-18a was nearly totallysuppressed (Fig. 3a, lane 4; see Supplementary Fig. 2 online formeasurements of hnRNP A1 depletion). This suggests an essential rolefor hnRNP A1 before or during the cropping of pri-miR-18a byDrosha. By contrast, depletion of hnRNP A1 had no impact on the
miR–18a*
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Figure 1 Identification of a binding site for hnRNP A1 in pre-miR-18a.
(a) Genomic structure of the C13orf25 gene, harboring the intronic
mir-17–92 cluster. Boxes, exons; lines, introns. Numbers below are those of
the miRNAs generated from each stem-loop structure. (b) Hairpin structure
of the precursor sequence for miR-18a and miR-18a*. Bold, CLIP tag that
bound hnRNP A1. Italic, sequences corresponding to the mature miRNAs
(miR-18a and miR-18a*).
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Figure 2 hnRNP A1 binds pri-miR-18a but not other pri-miRNAs in the same
cluster. (a) hnRNP A1 binds pri-miR-18a before cleavage by Drosha. After
immunoprecipitation with hnRNP A1–specific antibody (4B10), the bound
RNAs (lanes 1, 2, 5 and 6) or HeLa total RNA (B1/150 of the
immunoprecipitated RNA, lanes 3, 4, 7 and 8) were amplified by RT-PCR
with primers specific for the mir-17 cluster (lanes 1–4) or the mir-23 cluster
(lanes 5–8). M, 100-base-pair marker lane. (b) Agarose beads were mock-
treated (lane 2) or coupled to either pri17–19a (lane 3 and upper diagram)or pri23–24.2 (lane 4 and lower diagram). Bound proteins were analyzed by
western blotting with specific antibodies. In lane 1, 1/50 of the input
extract was loaded as control sample. (c) As in b, but with RNA
sequences corresponding either to only pre-miR-18a (pre18a) or to only
pre-miR19a (pre19a).
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efficient processing of the control pri23–24.2 substrate (comparelanes 6 and 7), indicating that the requirement for hnRNP A1 is aspecific feature of the mir-17 cluster.
We next wanted to investigate whether hnRNP A1 regulates only pri-miR-18a processing or whether it is necessary for maturation of theneighboring miRNAs. With that aim, we prepared two new RNA sub-strates, pri17–19afull (which comprises pri-miR-17, pri-miR-18a andpri-miR-19a plus B100 nucleotides on either end of the cluster) andpri17–92 (a 1.3-kilobase RNA substrate that encompasses the wholecluster sequence) (Fig. 3b, see diagram). Pre-miR-18a was the only pre-miRNA whose processing was reduced when it was incubated in extractsfrom cells depleted of hnRNP A1. Both the neighboring pre-miR-17 andpre-miR-19a and also the more downstream pre-miR-20a, pre-miR-
19b-1 and pre-miR-92 were insensitive to the reduced abundance ofhnRNP A1 (Fig. 3b; compare lanes 2 and 3, Supplementary Fig. 1).Moreover, addition of recombinant hnRNP A1 could rescue, althoughonly to a certain degree, the amount of pre-miR-18a in depleted extractsand could also increase its abundance in control extracts. (Fig. 3c, lanes3–5 and 7–9; compare with lanes 2 and 6, which contain no extraprotein). The limited effect of adding back hnRNP A1 is most probablydue to a partial loss of activity of the recombinant hnRNP A1 protein.Notably, however—and in contrast to the effect on pre-miR-18a—thisaddition did not alter the amount of processed pre-miR-17 and pre-miR-19a, reinforcing the specificity of hnRNP A1 function. Addition ofrecombinant SF2/ASF protein did not change the relative abundances ofpre-miRNAs and resulted only in a general repression of processing athigh concentrations either in control or hnRNP A1–depleted extracts(data not shown and Supplementary Fig. 3 online).
hnRNP A1 could be acting at the level of pre-miRNA stability,by preventing pre-miR-18a degradation, rather than enhancingthe cropping of pri-miR-18a. To test this possibility, we analyzedthe amounts of pre-miR-18a generated along the time courseof the in vitro reaction in extracts with diminished amounts ofhnRNP A1 (Supplementary Fig. 4a online). The abundanceof pre-miR-18a does not decrease with time, and although the kineticsof its production seem somewhat slower than those of pre-miR-17or pre-miR-19a, we conclude that its stability is not severely com-promised in the absence of hnRNP A1. In addition, in vitro–transcribed RNAs corresponding to the pre-miR-17, pre-miR-18a
Figure 3 hnRNP A1 facilitates Drosha-mediated
processing of miR-18a in vitro. (a) In vitro
processing of pri17–19a and pri23–24.2
(control cluster) in total or hnRNP A1–depleted
HeLa extracts. Primary RNA sequences shown
in diagrams were incubated with control extract
(lanes 2, 3 and 6) or hnRNP A1–depleted
extract (lanes 4 and 7) for the times indicated.
Products were analyzed on an 8% polyacrylamide
gel. Lanes 1 and 5, controls with no extract
added. M, RNA size marker. (b) hnRNP A1
functions specifically in processing of
pri-miR-18a. In vitro pri-miRNA processing
reactions were carried out with pri17–19afull or
with the whole pri17–92 cluster. These RNAswere incubated with total extracts (lane 2) or
hnRNP A1–depleted extracts (lane 3) and
analyzed as in a. Identities of labeled pri- and
pre-miRNA bands were confirmed by northern
blot analysis (see Supplementary Fig. 1).
(c) Recombinant hnRNP A1 can partially rescue
the defect in in vitro miR-18a processing.
Pri17–19afull was incubated with control extract
(lanes 2–5) or hnRNP A1–depleted extract (lanes 6–9) and in the absence (lanes 2 and 6) or presence of additional recombinant hnRNP A1 at a final
concentration of 4, 8 or 16 mg ml–1 (lanes 3 and 7, 4 and 8, and 5 and 9, respectively). Lane 1, control reaction in the absence of extract.
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Figure 4 Chimeric constructs reveal the influence of context in pre-miR-18a
production. (a–c) The chimeric substrates pri17-27-19a and pri23-18a-
24.2 (a) were tested with their wild-type counterparts in in vitro processing
assays. Each substrate was incubated in the absence of extract (lanes 1and 4), with control extracts (lanes 2 and 5) or with hnRNP A1–depleted
extracts (lanes 3 and 6). Identities of labeled pri- and pre-miRNA bands
were confirmed by northern blot analysis (see Supplementary Fig. 1).
(d) hnRNP A1 is not required for in vitro processing of pri106a–20b.
pri106a–20b was incubated in control HeLa extracts (lane 2) or hnRNP
A1–depleted extracts (lane 3). Lane 1, control reaction with no extract.
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and pre-miR-19a were equally stable when incubated in control orhnRNP A1–deficient extracts for increasing amounts of time under theconditions of in vitro processing reactions (Supplementary Fig. 4b).Together, these results indicate that hnRNP A1 has a specific role inregulating pri-miR-18a maturation at the level of primary miRNAprocessing by Drosha.
hnRNP A1’s role in processing is context dependentTo further investigate the mechanism by which this is achieved, wecreated chimeric constructs in which the pri-miR-18a and thepri-miR-27 sequences were swapped to generate the new substratespri17-27-19a and pri23-18a-24.2 (Fig. 4a). When placed betweenpri-miR-17 and pri-miR-19a, pri-miR-27 was efficiently processedregardless of the presence of hnRNP A1 (Fig. 4b, lanes 5 and 6).Moreover, in the new context of pri23-18a-24.2, we observed that theprocessing of pri-miR-18a was also unaffected by the presence ofhnRNP A1 (Fig. 4c, compare lanes 5 and 6). Notably, processing of thehighly homologous substrate pri106a–20b from the mir-106a clusteron chromosome X was not affected by hnRNP A1 depletion (seeFig. 4d and Supplementary Fig. 5 online). These data indicate thathnRNP A1 is required specifically for pri-miR-18a processing in acontext-dependent manner, suggesting the possibility that thesequence and natural context of pri-miR-18a constitute a suboptimalrecognition site for cropping by the Drosha–DGCR8 complex. As thefragment swapped between the constructs includes 30 nucleotides ateach side of pre-miR-18a, we hypothesize that sequences furtherupstream or downstream act in combination with hnRNP A1–bindingsites in the stem-loop to make the processing of pri-miR-18a topre-miR-18a dependent on hnRNP A1.
hnRNP A1 increases the processing of endogenous pri-miR-18aTo address the physiological relevance of hnRNP A1 regulation, theabundance of pre-miR-18a in the nucleus of control cells or cellsdepleted of hnRNP A1 by RNAi was measured by northern blotanalysis. We found that pre-miR-18a abundance was greatly reducedin HeLa cells depleted of hnRNP A1 (Fig. 5a, upper gel). The smalldifferences in size between the in vitro–generated pre-miRNAs (Fig. 3)
and those detected from in vivo samples by northern blotting (Fig. 5a)are at least in part due to the different types of polyacrylamide gel (8%in in vitro assays, 10% in northern blots), as demonstrated inSupplementary Figure 6 online. By contrast, according to the invitro data, the other members of the cluster showed no substantialreduction upon depletion of hnRNP A1 (Fig. 5a, middle and bottomgels, and Fig. 5b). The decrease in pre-miR-18a levels was con-comitant with an accumulation of pri-miR-18a, as detected byRT-PCR (Fig. 5c, left gel).
Finally, to test the implications at the level of mRNA targetregulation, we used a luciferase-based reporter to assay for miR-18aactivity. A target site for miR-18a was cloned downstream of the fireflyluciferase ORF, and the plasmid was transiently transfected into HeLacells (Fig. 5d). When overexpressed, miR-18a induces about a three-fold decrease in luciferase activity. By contrast, RNAi-mediateddepletion of hnRNP A1 resulted in derepression of the luciferasemRNA and a B2.5-fold increase in luciferase activity. Overexpressionof hnRNP A1 resulted in only a moderate increase in miR-18aabundance (Fig. 5e) and thus produced a weak repressive effect onluciferase expression (Fig. 5d). Together, these results indicatethat hnRNP A1 is required for miR-18a repression of a functionaltarget in vivo.
DISCUSSIONThe expression of mammalian miRNAs can be regulated at the post-transcriptional level by modulating nuclear and cytoplasmic miRNA-processing events. For instance, many miRNA transcripts are notprocessed by Drosha during early mouse development or duringcancer progression36. It has also been suggested that cleavage of pre-miRNAs in the cytoplasm by Dicer is regulated in such a way that thepresence of the functional mature miRNA is restricted to only afraction of the tissues where the pre-miRNA is initially expressed37.The mechanism by which this fine regulation of miRNA expression isachieved is not yet understood.
Here we have shown that hnRNP A1 can facilitate processing ofa particular miRNA in a context-dependent manner. Pri-miRNA
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Figure 5 hnRNP A1 increases the abundance of endogenous processed
miR-18a. (a) Northern blot of HeLa cells depleted of hnRNP A1 reveals a
reduction in pre-miR-18a. Nuclear RNA from control (lane 2) or hnRNP
A1–depleted cells (lane 1) was blotted with specific probes to detect
pre-miR-18a, pre-miR-20a and pre-miR-92. (b) As a loading control,
ethidium bromide stain of the RNA is shown. (c) Depletion of hnRNP A1
by RNAi results in the accumulation of pri-miR-18a. Total RNA from control
(lane 3) or depleted cells (lanes 1 and 2) was amplified with specific
primers (arrows in diagram) to detect the fragments corresponding to
pri-miR-18a (left gel) or pri-miR-20a (right gel). Two different hnRNP A1
sequences (A1-RNAi-1 and A1-RNAi-2; see Methods) were targeted by RNAi
to deplete hnRNP A1. (d) hnRNP A1 is required for miR-18a repression of a
luciferase indicator plasmid in vivo. HeLa cells were transfected with empty
plasmid (Empty) or plasmid encoding miR-18a, hnRNP A1 or a small
hairpin RNA to elicit RNAi-mediated depletion of hnRNP A1 (RNAi-A1).The cells were then retransfected with a firefly luciferase reporter
(Luc) whose 3¢ untranslated region contained a target site for miR-18a
(dark gray box in diagram), along with a Renilla luciferase (rLuc)
reporter (to control for transfection efficiency). SV40, simian virus 40
promoter; LCS, firefly luciferase open reading frame. A scrambled sequence
replacing the miR-18a target site was also tested as a control. Data shown
are means from three independent experiments; error bars show s.d.
(e) Abundance of miR-18a in cells transfected for experiments in d.
At left, 10 mg of total RNA from each sample analyzed on a 10% (w/v)
polyacrylamide gel and blotted with miR-18a–specific probe. At right, same
samples analyzed on a different gel and stained with ethidium bromide to
measure loading.
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maturation follows a two-step mechanism in which the stem-loopstructure and neighboring single-stranded RNA fragments are firstrecognized and tightly bound by DGCR8 at the junction between thesingle- and double-stranded RNA, then cropped by the recruitedDrosha35. The base of the stem-loop, as well as the structure of anyinternal loops, can influence both the efficiency and accuracy ofrecognition and processing. Our data show that hnRNP A1 facilitatesprocessing of miR-18a in a context-dependent manner, emphasizingthe importance of the pri-miRNA sequences surrounding miR-18a inthe requirement for hnRNP A1 (Fig. 4). In this regard, we canhypothesize that binding of hnRNP A1 to the stem-loop structurecould act in two ways: ensuring the maintenance of an optimalsecondary structure for DGCR8 and Drosha recognition and/orpreventing the binding of some unknown inhibitory factor(s).
In summary, a general RNA-binding protein that has been impli-cated in the regulation of alternative splicing has a role in theproduction of miR-18a. These results underscore a previously unchar-acterized role for RNA-binding proteins in the processing of specificmiRNAs. They also suggest the requirement of specific cofactors forthe processing of individual miRNAs, uncovering a new level ofcomplexity in the regulation of miRNA production and function.
METHODSPlasmids. All sequences used for in vitro transcription and primary miRNA
processing were cloned in pGEM-T-easy (Promega) and linearized with SpeI
before transcription. The DNA fragments were obtained by PCR from human
genomic DNA using the following oligonucleotide primers: for pri17–19a,
preclusterfor (5¢-CCAGTCAGAATAATGTCAAAGTGC-3¢) and 19rev (5¢-GCC
ACCATCAGTTTTGCATAG-3¢); for pri23–24.2, mir23for (5¢-CGCCCGGTGC
CCCCCTCACCCCTGTGCCAC-3¢) and miR24.2rev (5¢-CCCTGTTCCTGCT
GAACTGAGCCAGTGTAC-3¢); for pri17–19afull, 844for (5¢-GAATTCTTA
AGGCATAAATACG-3¢) and 1553rev (5¢-GTAGATAACTAAACACTACC-3¢);
for pri17–92, 675for (5¢-ACATGGACTAAATTGCCTTTAAATG-3¢) and 1991rev
(5¢-CCAAATCTGACACGCAACCCC-3¢); and for pri106a–20b, clusterXfor
(5¢-CAGGAATATTAACTAGTAG-3¢) and clusterXrev (5¢-ACGCTGAAATG
CAAACCTGC-3¢). The swapped constructs pri17-27-19a and pri23-18a-24.2
were derived from pri17–19a and pri23–24.2 as follows. The primary
sequences pri-miR-18a and pri-miR-27 were amplified by PCR with primers
clus17-103for (5¢-GTGCAGGGCCTGCTGATGTTGAGTGC-3¢) and clus17-
233rev (5¢-GAATTATTGGATGAATACATAAC-3¢), and primers clus23-143for
(5¢-GGCAGAGAGGCCCCGAAGCC-3¢) and clus23-279rev (5¢-CAGGCGGC
AAGGCCAGAGGAGG-3¢), respectively. These fragments were blunt-ligated
into new host plasmids from which the central pri-miRNA sequence had
previously been deleted. The deletion was done by PCR amplification with
primers clus23-141rev (5¢-CCCCGTCCCCGGGCAGCATCC-3¢) and clus23-
281for (5¢-CCCCTGCTGCCGCCTGTCTGCC-3¢), which render the fragment
pri23–24.2 with pri-miR-27 deleted; or with primers clus17-80rev (5¢-TAAAGCCCAACTTGGCTTCCCG-3¢) and clus17-236for (5¢-GCCAAGCAAG
TATATAGGTG-3¢), which render the fragment pri17–19a with pri-miR-18a
deleted. For the RNA pull-down experiments, the pre-miR-18a and pre-miR-
19a fragments were transcribed in vitro from a DNA fragment containing the
T7 promoter sequence directly upstream of the miRNA precursor sequence.
Similarly, the RNAs for the pre-miRNA stability assay were transcribed from
short PCR fragments using the following primers: for pre-miR-17, 5¢-TTAA
TACGACTCACTATAGGGCGTCAGAATAATGTCAAAGTGC-3¢ and 5¢-GTCA
CCATAATGCTACAAGTGCC-3¢; for pre-miR-18a, 5¢-TTAATACGACTCACTA
TAGGGCTGTTCTAAGGTGCATCTAGTGC-3¢ and 5¢-TGCCAGAAGGAGCA
CTTAGGGC-3¢; and for pre-miR-19a, 5¢-TTAATACGACTCACTATAGGGCG
CAGTCCTCTGTTAGTTTTGC-3¢ and 5¢-GCAGGCCACCATCAGTTTTGC-3¢.For overexpression of hnRNP A1, we used the mammalian expression plasmid
pCGT-A1, which carries a T7-tagged version of hnRNP A1 (ref. 38). Knock-
down of hnRNP A1 was achieved by transfection of the pSUPER plasmid
(Oligoengine), encoding a small hairpin RNA targeting the sequence 5¢-AGCA
AGAGATGGCTAGTGC-3¢ (A1-RNAi-1) or 5¢-TGAGAGATCCAAACACCAA-3¢(A1-RNAi-2), as described39. The sequence A1-RNAi-1 was the one used for
depletion of hnRNP A1 in all the experiments except that in Figure 5c, where
both sequences, A1-RNAi-1 and A1-RNAi-2, were tested. The luciferase
reporters were constructed as follows. The target site for miR-18a was cloned
into the NotI and PstI sites of pBPLUGA40 with oligonucleotides 5¢-GATCT
TATCTGCACTAGATGCACCTTACTGCA-3¢ and 5¢-GTAAGGTGCATCTAGT
GCAGATA-3¢. The control scrambled sequence was cloned using oligonucleo-
tides 5¢-GATCTATGGCACAATGCGGAGCTACCTCTGCA-3¢ and 5¢-GAGG
TAGCTCCGCATTGTGCCATA-3¢.
Cross-linking and immunoprecipitation. HeLa cells were cross-linked in vivo,
and the RNA-binding targets for hnRNP A1 were obtained as described20,21,
with the following modifications. In brief, 10-cm dishes were cross-linked at
400 mJ cm–2, cells were collected and nuclear extracts were prepared in lysis
buffer (1� PBS, 0.125% (w/v) SDS, 0.5% (w/v) deoxycholate, 0.5% (v/v)
Nonidet P-40). After DNase treatment and a mild digestion with RNase A and
RNase T1, the extracts were immunoprecipitated for 1 h at 4 1C with the
monoclonal antibody 4B10 (Immuquest) coupled to protein A–Sepharose
beads (Roche). After extensive washing with lysis buffer and PNK buffer
(50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 0.5% (v/v) Nonidet P-40), the
cross-linked RNAs were dephosphorylated and ligated overnight to the 3¢ RNA
linker. After 5¢ end labeling, the samples were loaded onto a 10% (w/v) Bis-Tris
gel (Novex NuPAGE) and the appropriate shifted protein bands were cut out of
the gel. The RNA was purified, ligated to the 5¢ linker and amplified by RT-PCR
as described before.
Immunoprecipitation and reverse-transcription PCR. Endogenous hnRNP
A1 was immunoprecipitated from total HeLa extracts as described41. The
pulled-down RNA was isolated with TRI reagent (Sigma) and reverse tran-
scribed using Superscript II (Invitrogen) with the specific primer preclusterrev
(5¢-CAGTGGAAGTCGAAATCTTCAG-3¢) for pri17–92 and mir24 cDNA
(5¢-GGCAGGGGCTGCAGGCTCCAAGGGGGCTTG-3¢) for pri23–24.2. For
PCR amplification, primers 17dir (5¢-CAAAGTGCTTACAGTGCAGG-3¢) and
preclusterrev were used for pri17–92, and primers miR23for (5¢-CGCC
CGGTGCCCCCCTCACCCCTGTGCCAC-3¢) and miR24.2rev (5¢-CCCTGT
TCCTGCTGAACTGAGCCAGTGTAC-3¢) were used for pri23–24.2. Pri-miR-
NAs were detected by RT-PCR using the Superscript III One-Step RT-PCR
System with Platinum Taq High Fidelity (Invitrogen), with oligonucleotides
5¢-GTGCAGGGCCTGCTGATGTTGAGTGC-3¢ and 5¢-GAATTATTGGATGA
ATACATAAC-3¢ for pri-miR-18a, and 5¢-GTGTCGATGTAGAATCTGCC-3¢and 5¢-GCAGTACTTTAAGTGCTCAT-3¢ for pri-miR-20a.
In vitro pri-miRNA processing assays. Total HeLa extracts were prepared from
B3 � 106 control or hnRNP A1–depleted cells resuspended in 1 ml of buffer D
(20 mM HEPES-KOH (pH 7.9), 100 mM KCl, 0.2 mM EDTA, 0.5 mM DTT,
0.2 mM PMSF, 5% (w/v) glycerol). The suspension was sonicated and
centrifuged for 5 min at 10,000g, and the supernatant used for in vitro assays.
Pri-miRNA substrates were prepared by standard in vitro transcription with
T7 RNA polymerase in the presence of [a-32P]GTP. Before transcription,
template DNAs were linearized with SpeI. Assays were done in 30-ml reaction
mixtures containing 50% (v/v) total or depleted HeLa extract, 0.5 mM ATP, 20
mM creatine phosphate, 3.2 mM MgCl2 and 20,000 c.p.m. (B10 fmol) of each
pri-miRNA. Reactions were incubated at 30 1C for 90 min, then subjected
to phenol-chloroform extraction, precipitation and 8% (w/v) denaturing
gel electrophoresis.
RNA affinity purification and western blotting. Substrate RNAs for bead
immobilization were synthesized in vitro from plasmids pGEM-T-easy-
pri17–19a and pGEM-T-easy-pri23–24.2 (linearized with SpeI), or from the
DNA templates T7-pre18a and T7-pre19a, using T7 RNA polymerase. After
transcription, all RNAs were gel-purified. Coupling to beads and affinity
purification of factors were done as described11. Proteins were separated on
10% (w/v) SDS-polyacrylamide gels and immunoblotted with hnRNP A1–
specific monoclonal antibody 4B10 (Abcam), with SF2/ASF-specific mono-
clonal antibody (no. 96; see Acknowledgments)42 or with hnRNP K/J–specific
monoclonal antibody 3C2 (Immuquest).
Northern blots. Total nuclear RNA (B5 mg) was resolved on a 10% (w/v)
denaturing polyacrylamide gel (7 M urea) and transferred to Hybond N
membranes (Amersham Pharmacia Biotech), with the exception of the blot
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in Supplementary Figure 6, where an 8% (w/v) denaturing gel (8 M urea) was
used. The membranes were then prehybridized for 4 h at 45 1C in 1� SSC,
1% (w/v) SDS and 100 mg ml–1 single-stranded DNA (Sigma). Probes
corresponding to the stem-loop of pre-miR-18a, pre-miR-20a or pre-miR-92
were synthesized using the mirVana miRNA Probe Construction Kit (Ambion)
and left to hybridize overnight in 1� SSC, 1% (w/v) SDS and 200 mg ml–1
single-stranded DNA. After hybridization, the membranes were washed four
times at 50 1C in 0.2� SSC and 0.2% (w/v) SDS for 30 min each. The blots
were then analyzed using a PhosphorImager (Molecular Dynamics).
Cell transfection and dual luciferase assays. HeLa cells were grown to 80%
confluence in 24-well plates. Knockdown of hnRNP A1 was achieved by
transfection of the pSUPER plasmid (Oligoengine), encoding a small hairpin
RNA targeting the sequence 5¢-AGCAAGAGATGGCTAGTGC-3¢, with Lipo-
fectamine 2000 (Invitrogen), following the manufacturers’ instructions. For
hnRNP A1 or miR-18a overexpression, pSUPER-miR-18a or T7-hnRNP A1
plasmids were transfected in a similar way. The transfection medium was
replaced with fresh medium after a 5-h incubation, and cells were then
incubated for another 24 h. Cells were then retransfected with the firefly
luciferase reporters, along with the Renilla luciferase transfection control. For
dual luciferase assays HeLa cells were lysed using passive lysis buffer (Promega),
and the levels of firefly and Renilla luciferase activity were measured using the
Promega Dual Luciferase Reaction system. The data are expressed as a ratio of
firefly luciferase activity to Renilla luciferase activity. The luminescence was
measured with a Monolight 3010 luminometer (Pharmingen).
Note: Supplementary information is available on the Nature Structural & MolecularBiology website.
ACKNOWLEDGMENTSWe thank N. Hastie for comments and critical reading of the manuscript, andA. Krainer (Cold Spring Harbor Laboratory) for the generous gift of SF2/ASFantibody and recombinant hnRNP A1 protein. This work was supported by theMedical Research Council and Eurasnet (European Alternative Splicing Network-FP6). S.G. was supported by a European Molecular Biology Organization long-term postdoctoral fellowship.
AUTHOR CONTRIBUTIONSS.G. and J.F.C. conceived, designed, and interpreted the experiments. S.G.performed the experiments and data analysis. J.F.C. supervised the whole project.The manuscript was cowritten by both authors.
COMPETING INTERESTS STATEMENTThe authors declare no competing financial interests.
Published online at http://www.nature.com/nsmb/
Reprints and permissions information is available online at http://npg.nature.com/
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