[RNA Biology 3:2, 1-1, EPUB Ahead of Print: http://www.landesbioscience.com/journals/rnabiology/abstract.php?id=3110; April/May/June 2006]; ©2006 Landes Bioscience

1 RNA Biology 2006; Vol. 3 Issue 2

Guy A. Tremblay†

Stéphane Richard*

Terry Fox Molecular Oncology Group and Bloomfield Center for Research on Aging;Lady Davis Institute for Medical Research; Quebec Canada

Departments of Oncology and Medicine; McGill University; Montreal, QuebecCanada

†Present address: INRS-Institut Armand-Frappier; Université de Montréal; Laval,Québec Canada

*Correspondence to: Stéphane Richard; Lady Davis Institute; 3755 Côte Ste.-Catherine Road; Montréal, Québec H3T 1E2 Canada. Tel.: 514.340.8260; Fax:514.340.8295; Email: stephane.richard@mcgill.ca

Received 05/17/06; Accepted 07/15/06

This manuscript has been published online, prior to printing for RNA Biology,Volume 3, Issue 2. Definitive page numbers have not been assigned. The current cita-tion is: RNA Biology 2005; 3(2):http://www.landesbioscience.com/journals/rnabiology/abstract.php?id=3110Once the issue is complete and page numbers have been assigned, the citation willchange accordingly.

KEY WORDS

Src associated substrate in mitosis of 68kDa,Sam68, KH domain, RNA metabolism, mRNAtargets

ACKNOWLEDGEMENTS

This work was funded by grant MT-13377 fromthe Canadian Institutes of Health Research(CIHR). S.R. is an investigator of the CIHR.

Research Paper

mRNAs Associated with the Sam68 RNA Binding Protein

ABSTRACTThe Src associated substrate in mitosis of 68kDa, Sam68, is an RNA-binding protein

that belongs to the KH domain family of proteins. KH-type RNA binding proteins areknown to mediate high affinity RNA binding and regulate RNA metabolism includingpre-mRNA splicing, mRNA export and protein translation. The RNA binding specificity ofSam68 as well as its RNA targets are poorly understood. Herein we cross-linked mRNAassociated with Sam68 and identified some of the mRNA associated with the Sam68RNA binding protein complex. By using this strategy, we have identified 23 mRNAs thatare associated with the immunoprecipitated endogenous Sam68 protein complex. Five ofthe identified mRNAs were validated by co-immunoprecipitation assay followed byreverse transcription PCR confirming that we had indeed identified mRNAs associatedwith the Sam68 protein complex.

INTRODUCTIONThe Src substrate associated in mitosis of 68 kDa (Sam68) is a known substrate of

several tyrosine kinases including Src family kinases and the BRK breast tumor kinase.1

The association of Sam68 with Src and BRK tyrosine kinases is mediated by SH3 and SH2domain-dependent interactions. Moreover, the phosphorylation of Sam68 by BRK wasshown to occur downstream of the epidermal growth factor in breast tumor cell lines.2

Sam68 has been shown to serve several cellular roles including mRNA splicing3 and viralRNA export.4 The physiological role of Sam68 has remained elusive until recently. Sam68was shown to play a role in bone marrow mesenchymal stem cell differentiation, as micenull for Sam68 have an increased osteogenic differentiation.5

Sam68 is a known RNA binding protein that was initially shown to bind DNA andcellular RNA in vitro.6 It was then shown that Sam68 bound poly U7 and poly A ribonu-cleotide homopolymer.8 It was later demonstrated by using SELEX (systematic evolutionof ligands by exponential enrichment) with bacterial recombinant protein that Sam68bound RNAs containing UAAA and UUUA motifs with high affinity.9 By using bacteriallyexpressed glutathione-S-transferase Sam68 fusion proteins immobilized on glutathionebeads bound cellular mRNAs were identified by differential display and cDNA-RDA. TenmRNAs were validated in HeLa cells over-expressing HA-Sam68.10 Abundant mRNAswere identified including hnRNP A2/B1 and β-actin, whose binding sites were mapped toUAAA and UUUUUU consistent with the previous homopolymeric RNA and SELEXdata. The difficulty with the interpretation of these previous experiments is that bacteriallypurified Sam68 was utilized and since Sam68 is extensively modified post-transcription-ally,1,11-13 the recombinant protein may not properly reflect the specific RNA bindingactivity of the post-translationally modified protein. Therefore the physiological mRNAtargets will best be identified using the purified endogenous Sam68 complex frommammalian cells. The identification of RNA targets of Sam68 in vivo will help unravelthe cellular function of Sam68 and find its binding sites on RNAs. Bearing this in mind,we decided to identify the mRNAs bound to Sam68 in vivo. Herein, we identify mRNAsbound to the endogenous Sam68 complex using NIH 3T3 cells that were cross-linkedwith UV light. Subsequently, the bound mRNAs were amplified by using an oligo dTbased approach and the cDNAs cloned. We identified 23 mRNA targets bound to theSam68 complex that were absent in the mock immunoprecipitations. We validated fivemRNA targets by immunoprecipitation reverse transcription PCR demonstrating thatthese mRNAs are associated with the Sam68 RNA binding protein complex. The challengewill be to identify the mRNAs that specifically associate with Sam68.

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MATERIALS AND METHODSUV cross-linking and immunoprecipitations. NIH 3T3 grown to

near confluence in 10 cm tissue culture petri dishes were washedwith 1X phosphate buffered saline (PBS) and UV cross-linked with120,000 microjoules/cm2 in a Stratalinker (Stratagene Inc.). Thecells were subsequently lysed for 10 min in lysis buffer (1% TritonX-100, 150 mM NaCl, 20mM Tris pH7.4, 1 mM PMSF), and thecellular debris removed by centrifugation. The supernatant wasimmunoprecipitated with either normal rabbit serum (NRS) or withanti-Sam68 antibodies.14 The immune complexes were immunopre-cipitated for one hour using Dynabeads® Protein A (DynalBiotechnology Inc.) supplemented with the following RNaseinhibitors:10 mM Ribonucleoside Vanadyl Complex (New EnglandBiolabs Inc.) and 200 units SUPERase-In (Ambion Inc.) in thepresence of 1 mg/ml Heparin and 20 µg/ml yeast tRNA were addedas blocking reagents in binding and lysis reactions. The beads werewashed thoroughly with lysis buffer and subsequently washed fourtimes with PBS containing 0.1% Triton X-100.

Converting bound mRNAs into cDNAs. The first strand synthesiswas performed while the Sam68 immune complex was bound to theDynabeads®. Reverse transcription was performed with an oligo dToligonucleotides with a 3' anchor (underlined): 5'-GGG AGA CAAGAA TAA ACG CTC AAT TTT TTT TTT TTT TTT TTT TVN-3' (V represents equal amounts of A, C and G; N represents anynucleotide) and the Superscript II Reverse Transcriptase (RNase Hminus; Invitrogen Inc.) for one hour and then the beads werewashed with PBS:0.1% Triton X-100. The second-strand synthesiswas performed by the addition of DNA polymerase I (New EnglandBiolabs Inc.), E. coli RNase H (Invitrogen Inc.) and E. coli DNAligase (New England Biolabs Inc.) at 16˚C for two hours, asdescribed previously.15

Amplification of cDNAs and their identification. The cDNAsgenerated were amplified by linear PCR (with one primer) using0.00625 units/µl Taq DNA polymerase (Promega Inc.), 1.5 mMMgCl2, 20 µCi dCTP α32P and 1.25 mM of the anchor primer(5'-GGG AGA CAA GAA TAA ACG CTC AA-3'). The conditionswere: 95˚C/20 s; 53˚C/20 s; 72˚C/3 min for 35 cycles. The singlestranded DNA fragments over 500 nucleotides were gel extracted froma denaturing 7M urea / 5% polyacrylamide gel. An RNA ligationprimer with its 3' hydroxyl group blocked with an amino modificationto prevent 3' ligation (5'-PO4-CGA GAU GGC GGC UUC CUGC-blocked 3') was ligated at the 3' end of the amplified cDNAs withT4 RNA ligase (Fermentas Inc.). Finally, the DNA fragments wereamplified by PCR using primers that complement the ligation andanchor primers, but that introduce flanking EcoRI and BamHI sitesfor subloning in Bluescript KS to facilitate DNA sequencing atGenome Centre (McGill University). In a second strategy, a DNAligation primer that complements the anchor (5'-PO4-TTG AGCGTT TAT TCT TGT CTC CC-blocked 3') was utilized with T4DNA ligase to ligate at the 3' end of the amplified cDNAs. TheDNA inserts were amplified by using suppression PCR with oneprimer that introduces flanking EcoRI sites.

Validation of mRNAs associated with the Sam68 complex.Specific mRNAs were isolated using TRIzol (Life Technologies Inc.)following control or anti-Sam68 immunoprecipitations as describedpreviously.16 Reverse transcription with the oligo dT primer wasperformed followed by 25 to 35 cycles of PCR with the designatedprimers as follows: GAPDH: 5'-TGC TGA GTA TGT CGT GGAGTC T-3' and 5'-ATC ATA CTT GGC AGG TTT CTC C-3',

FBP11: 5'-CGG AAA GAG TCT GCC TTT AAG A-3' and 5'-TTT TCA CTG TCC CGA TCT TTT T-3', myl6: 5'-CCC CAAGAG TGA TGA GAT GAA T-3' and 5'-ATT TTA TTT GGGGAA GGC AAA C-3', Ena/Vasp: 5'-CAT GGA AGA AAT GAACAA GCT G-3' and 5'-CCA GGA GTT GAA GTT TGT TTC C-3', prof1: 5'-CCA TCG TAG GCT ACA AGG ACT C-3' and 5'-AAT AAG GGA AAT GGG GTA ATG G-3', thrap2: 5'-GTG ACTTGA GCC AAT GTG TGA T-3' and 5'-TTC TCA CTC CTTTCT TGG CTT C-3', copeb: 5'-CGA CCA AAT TTA CCT CTGATC C-3' and 5'-TTA AAA GGC TTG GCA CCA GTA T-3',Tctp: 5'-AGG GCA AGA TGG TCA GTA GAA C-3' and 5'-ATGCCA CCA CTC CAA ATA AAT C-3', ≤-actin: 5'-CCT GAA GTACCC CAT TGA ACA T-3' and 5'-CTG CTC GAA GTC TAGAGC AAC A-3'. All oligonucleotides utilized above for the RT-PCRvalidation yield DNA fragments of 400-600 base pairs in length thatwere separated on 2% agarose gels and visualized by ethidiumbromide staining.

RESULTSIdentification of mRNA targets bound to the Sam68 complex.

To identify the mRNAs bound to endogenous Sam68, we cross-linked asynchronous NIH 3T3 cells, a cell type known to expressabundant levels of Sam68, with ultraviolet irradiation to ensure thecovalent association of the mRNAs with the Sam68 complex. TheUV cross-linking allows the preservation of the Sam68 ribonucleo-protein (RNP) complex during the immunoprecipitation. Thebound mRNAs were converted into cDNAs by reverse transcription.

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Figure 1. Visualization of the amplified cDNAs bound to the Sam68 com-plex. Lanes 1-3, suppression PCR strategy. Lane 1, no template control (-DNA). Lane 2, mock control immunoprecipitation (IP:) with normal rabbitserum (NRS). Lane 3, anti -Sam68 immunoprecipitation with AD1 antibody.Lanes 4-6, typical double primer PCR approach (regular PCR). Lane 4, notemplate control. Lane 5, mock control immunoprecipitation, lane 6, anti-Sam68 immunoprecipitation. The 'markers' indicates 1kb DNA ladder(Invitrogen).

Half the eluted cDNAs were amplified by suppression PCR to favorlonger DNA fragments (Fig. 1, lanes 1–3) and the other half wasamplified by regular 2 primer PCR (Fig. 1, lanes 4–6). The DNAfragments were purified, subcloned and sequenced (Table 1). TheGenbank accession number, the portion of the mRNA amplified,the redundancy of each clone is shown, and whether the polyA tailwas primed are shown. Surprisingly, no intronic sequences werefound, suggesting that the captured molecules corresponded indeedto mature mRNAs, as predicted for oligo dT primed reactions.However, the high level of internal priming of the oligo dT primers(Table 1) should have identified some pre-mRNA species associatedwith Sam68. Most likely the method was not sensitive enough or toofew clones were sequenced. The non-specific mRNAs associatedwith the mock immunoprecipitations were also cloned and weretypically tRNA, ribosomal protein genes and mitochondrial geneproducts (data not shown). In the clones identified with anti-Sam68immunoprecipitations ~20% represented non-specific RNAs andthe remaining were deemed mRNAs associated with the Sam68complex. A total of 23 mRNAs was captured with our analysis(Table 1).

Validation of mRNA targets bound to the Sam68 complex. Weproceeded with the validation of the Sam68 captured mRNA targets.NIH 3T3 cells were lysed and immunoprecipitated with eithercontrol normal rabbit serum (Mock) or the anti-Sam68 antibodies.The co-precipitating mRNAs were isolated and amplified withprimer sets corresponding to a chosen set of mRNAs from Table 1.We used β-actin as a positive control, since it a known target ofSam68,10,17 however we did not identify this mRNA in our assaydemonstrating that we have only identified a subset of Sam68associated mRNAs. As a negative control gene we used the glycer-aldehyde-3-phosphate dehydrogenase mRNA (GAPDH). It wasnegative, but we increased the number of PCR cycles until we coulddetect an equal amount of intrinsic noise between the mock and theanti-Sam68 antibody immunoprecipitations. Myl6, Copeb, Fnbp3,prof1 and Tctp were true positives and Ena/vasp and thrap2 may befalse positives as we could detect them in the corresponding mock IPlanes (Fig. 2). These data demonstrate that ~70% of the identifiedmRNAs are indeed true positives.

DISCUSSIONWe have identified 23 mRNAs bound to the endogenous Sam68

protein complex in NIH3T3 cells and all these contain UAAA,UUUA or poly(U) motifs. We believe that a subset of mRNAs wereidentified as positive controls such as β-actin were not identified inour assay. Furthermore, a correlation could not be made between theposition where reverse transcription reaction stopped, and a putativeSam68 binding site. There was one exception: the 5' end of thesequence for thrap2 mRNA, where the reverse transcription reactionstopped, is a poly(U) stretch and 2 UUUA motifs. It remains to bedetermined whether Sam68 binds a rather undefined site onmRNAs, and thereby a very large set of mRNA, or whether itrequires a specific cellular context in order to bind to its RNA targets,or any other reason thereof; one appealing theory involves mRNAtargets bound to other RNA binding proteins in the large Sam68complex. This latter possibility explains why a specific Sam68binding site cannot be identified with our approach.

The mRNAs identified in Table 1 can be functionally classifiedinto cell motility and migration (Prof1, Myl6, Evl, Fnbp3 and Bgn),transcription-related genes (Thrap2, Copeb, Hmgn1, Hmgb1 andBclaf1), cell cycling/cell division (Apc5, Smc6 and Nap1l1) and celldeath (Sesn2, Anx11, Bri3bp and Bclaf1). As phosphorylation ofSam68 by Src is known to negatively regulate RNA binding activity,18

and that Src will function to increase the motility and migration ofcancer cells, it follows that Sam68 should have a regulator effect onits mRNA targets, or at least those related to cell motility and migra-tion. Therefore we could speculate that tyrosine phosphorylation ofSam68 could liberate the mRNA sequestered by Sam68, renderingthem available for translation, for example. Recent observations byParonetto et al., suggest that shuttling of Sam68 from the nucleus tothe cytoplasm in spermatocytes correlates with phosphorylation, thatit associates with polysomes and may facilitate translation.19

In conclusion, we have shown that specific mRNAs can be capturedto the Sam68 RNA binding protein complex. We have defined asubset of the mRNAs that the Sam68 complex associates withmRNAs regulating cell mobility and migration, cell cycle and tran-scriptional regulation. Further studies are required to identify thecomplete list of Sam68 RNA targets and to define the specific bindingsites in the presence or absence of certain cellular signals.

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3 RNA Biology 2006; Vol. 3 Issue 2

Figure 2. Association of mRNAs with the endogenous Sam68 complex inNIH 3T3 cells. NIH3T3 cells were immunoprecipitated (IP) with either nor-mal rabbit serum (NRS) or rabbit polyclonal anti-Sam68 AD-1 antibody (±-Sam68). The co-immunoprecipitating mRNAs were subjected to reverse tran-scription PCR. Various sets of primers were used based on the mRNA targetsfrom Table I. GAPDH served as a negative control and ≤-actin served as apositive control. The 'markers' indicates 1kb DNA ladder (Invitrogen).

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breast tumor kinase regulates intranuclear localization and cell cycle progression. J BiolChem 2005; 280:38639-47.

3. Matter N, Herrlich P, Konig H. Signal-dependent regulation of splicing via phosphoryla-tion of Sam68. Nature 2002; 420: 691-5.

4. Reddy TR, Xu W, Mau JKL, Goodwin CD, Suhasini M, Tang H, Frimpong K, Rose DW,Wong-Staal F. Inhibition of HIV replication by dominant negative mutants of Sam68, afunctional homolog of HIV-1 Rev. Nature Medicine 1999; 5:635-642.

5. Richard S, Torabi N, Franco GV, Tremblay GA, Chen T, Vogel G, Morel M, Cléroux P,Forget-Richard A, Komarova S, Tremblay ML, Li W, Li A., Gao YT, Henderson JE.Ablation of the Sam68 RNA Binding Protein Protects Mice from Age-Related Bone Loss.PLoS Genet 2005; 1; e74.

6. Wong, G. et al. Molecular cloning and nucleic acid binding properties of the GAP-associ-ated tyrosine phosphoprotein p62. Cell 1992; 69; 551-558.

7. Taylor SJ, Shalloway D. An RNA-binding protein associated with src through its SH2 andSH3 domains in mitosis. Nature 1994; 368: 867-871.

8. Chen T, Damaj BB, Herrera C, Lasko P, Richard S. Self-association of the single-KH-domain family members Sam68, GRP33, GLD-1, and Qk1: role of the KH domain. MolCell Biol 1997; 17:5707-18.

9. Lin Q, Taylor SJ, Shalloway D. Specificity and determinants of Sam68 RNA binding. J.Biol. Chem 1997; 272: 27274-80.

10. Itoh M, Haga I, Li Q-H, Fujisawa J-I. Identification of cellular mRNA targets for RNA-binding protein Sam68. Nucl Acids Res 2002; 30: 5452-64.

11. Côté J, Boisvert FM, Boulanger MC, Bedford MT, Richard S. Sam68 RNA binding pro-tein is an in vivo substrate for protein arginine N-methyltransferase 1. Mol Biol Cell 2003;14: 274-87.

12. Babic I, Cherry E, Fujita DJ. SUMO modification of Sam68 enhances its ability to represscyclin D1 expression and inhibits its ability to induce apoptosis. Oncogene 2002; [Epubahead of print].

13. Babic I, Jakymiw A, Fujita DJ. The RNA binding protein Sam68 is acetylated in tumor celllines, and its acetylation correlates with enhanced RNA binding activity. Oncogene 2004;23:3781-9.

14. Chen T, Boisvert FM, Bazett-Jones DP, Richard, S. A role for the GSG domain in localiz-ing Sam68 to novel nuclear structures in cancer cell lines. Mol Biol Cell 1999; 10:3015-33.

15. Gubler U, Hoffman BJ. A simple and very efficient method for generating cDNA libraries.Gene 1983; 25: 263-9.

16. Galarneau A, Richard S. Target RNA motif and target mRNAs of the Quaking STAR pro-tein. Nat Struct Mol Biol 2005; 12:691-8.

17. Hill MA, Schedlich L, Gunning P. Serum-induced signal transduction determines theperipheral location of ≤-actin mRNA within the cell. J. Cell Biol 1994; 126: 1221-30.

18. Wang LL, Richard S, Shaw AS. p62 association with RNA is regulated by tyrosine phos-phorylation. J. Biol. Chem 1995; 270:2010-3.

19. Paronetto MP, Zalfa F, Botti F, Geremia R, Bagni C, and Sette C. The nuclear RNA-bind-ing protein Sam68 translocates to the cytoplasm and associates with the polysomes inmouse spermatocytes. Mol Biol Cell 2006; 17:14-24.

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TABLE 1 mRNA identified associated with the endogenous Sam68 protein complex in NIH3T3 cells

mRNA captured Accession number Position cloned Redundancy in CART polyA primed

thyroid hormone receptor associated protein 2 (Thrap2) BC040283 3776–4265 4 yesbri3 binding protein (bri3bp) XM_132386 931–1180 3 nonon-muscle myosin light chain 3 (Myl6) U04443 114–606 3 yescore promoter element binding protein (Copeb/KLF6/Zf9) NM_011803 998–1386 2 nohigh mobility group nucleosomal binding domain 1 (Hmgn1) NM_008251 499–962 2 nostructural maintenance of chromosome 6 (Smc6) AJ310552 1368–1870 2 nobiglycan (Bgn) BC052857 1908–2375 1 yesEna-VASP like protein (Evl) U72519 1702–1788 1 yesformin binding protein 3 (Fnbp3) NM_018785 1970–2348 1 noprofilin 1 (Prof1) BC002080 317–756 1 nohigh mobility group box 1 BC008565 410–654 1 nolipoprotein receptor-related protein (Lrp1) AF367720 14443–14783 1 yesnucleosome assembly protein 1-like 1 (Nap1l1) NM_015781 1564–1795 1 noanaphase promoting complex subunit 5 (APC5) BC046804 1917–2386 1 yescDNA coding for a hypothetical protein BC033455 857–1290 1 nocDNA coding for a hypothetical protein AY061989 1781–2312 1 nocDNA coding for a hypothetical protein NM_133718 3396–3011 1 nosestrin 2 (Sesn2) NM_144907 2251–2533 1 noannexin A11 (Anx11) BC012875 2040–2186 1 noBCL2-associated transcription factor 1 (BCLAF1) BC034300 2387–2838 1 notumor protein, translationally-controlled 1 (Tctp) NM_009429 395–722 1 nopoly A binding protein 1 (pabc1) BC011207 2034–2214 1 yesChr 10, ERATO Doi 214, expressed BC021952 200–408 1 no