[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
mRNAs Associated with the Sam68 RNA Binding Protein
Research Paper
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, Quebec
Canada
†Presentaddress: 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=3110
Once the issue is complete and page numbers have been assigned, the citation will
change accordingly.
KEY WORDS
Src associated substrate in mitosis of 68kDa,
Sam68, KH domain, RNA metabolism, mRNA
targets
ACKNOWLEDGEMENTS
This work was funded by grant MT-13377 from
the Canadian Institutes of Health Research
(CIHR). S.R. is an investigator of the CIHR.
1 RNA Biology
ABSTRACT
The 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 are
known to mediate high affinity RNA binding and regulate RNA metabolism including
pre-mRNA splicing, mRNA export and protein translation. The RNA binding specificity of
Sam68 as well as its RNA targets are poorly understood. Herein we cross-linked mRNA
associated with Sam68 and identified some of the mRNA associated with the Sam68
RNA binding protein complex. By using this strategy, we have identified 23 mRNAs that
are associated with the immunoprecipitated endogenous Sam68 protein complex. Five of
the identified mRNAs were validated by co-immunoprecipitation assay followed by
reverse transcription PCR confirming that we had indeed identified mRNAs associated
with the Sam68 protein complex.
INTRODUCTION
The 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 SH2
domain-dependent interactions. Moreover, the phosphorylation of Sam68 by BRK was
shown 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 viral
RNA export.4 The physiological role of Sam68 has remained elusive until recently. Sam68
was shown to play a role in bone marrow mesenchymal stem cell differentiation, as mice
null for Sam68 have an increased osteogenic differentiation.5
Sam68 is a known RNA binding protein that was initially shown to bind DNA and
cellular 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 evolution
of ligands by exponential enrichment) with bacterial recombinant protein that Sam68
bound RNAs containing UAAA and UUUA motifs with high affinity.9 By using bacterially
expressed glutathione-S-transferase Sam68 fusion proteins immobilized on glutathione
beads bound cellular mRNAs were identified by differential display and cDNA-RDA. Ten
mRNAs were validated in HeLa cells over-expressing HA-Sam68.10 Abundant mRNAs
were identified including hnRNP A2/B1 and β-actin, whose binding sites were mapped to
UAAA and UUUUUU consistent with the previous homopolymeric RNA and SELEX
data. The difficulty with the interpretation of these previous experiments is that bacterially
purified 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 binding
activity of the post-translationally modified protein. Therefore the physiological mRNA
targets will best be identified using the purified endogenous Sam68 complex from
mammalian cells. The identification of RNA targets of Sam68 in vivo will help unravel
the 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 mRNAs
bound to the endogenous Sam68 complex using NIH 3T3 cells that were cross-linked
with UV light. Subsequently, the bound mRNAs were amplified by using an oligo dT
based approach and the cDNAs cloned. We identified 23 mRNA targets bound to the
Sam68 complex that were absent in the mock immunoprecipitations. We validated five
mRNA targets by immunoprecipitation reverse transcription PCR demonstrating that
these mRNAs are associated with the Sam68 RNA binding protein complex. The challenge
will be to identify the mRNAs that specifically associate with Sam68.
2006; Vol. 3 Issue 2
 AUTHOR PLEASE PROVIDE RUNNING TITLE

MATERIALS AND METHODS

UV cross-linking and immunoprecipitations. NIH 3T3 grown to
near confluence in 10 cm tissue culture petri dishes were washed
with 1X phosphate buffered saline (PBS) and UV cross-linked with
120,000 microjoules/cm2 in a Stratalinker (Stratagene Inc.). The
cells were subsequently lysed for 10 min in lysis buffer (1% Triton
X-100, 150 mM NaCl, 20mM Tris pH7.4, 1 mM PMSF), and the
cellular debris removed by centrifugation. The supernatant was
immunoprecipitated with either normal rabbit serum (NRS) or with
anti-Sam68 antibodies.14 The immune complexes were immunopre-
cipitated for one hour using Dynabeads® Protein A (Dynal
Biotechnology Inc.) supplemented with the following RNase
inhibitors:10 mM Ribonucleoside Vanadyl Complex (New England
Biolabs Inc.) and 200 units SUPERase-In (Ambion Inc.) in the
presence of 1 mg/ml Heparin and 20 µg/ml yeast tRNA were added
as blocking reagents in binding and lysis reactions. The beads were
washed thoroughly with lysis buffer and subsequently washed four
times with PBS containing 0.1% Triton X-100.
Converting bound mRNAs into cDNAs. The first strand synthesis
was performed while the Sam68 immune complex was bound to the
Dynabeads®. Reverse transcription was performed with an oligo dT
oligonucleotides with a 3' anchor (underlined): 5'-GGG AGA CAA
GAA TAA ACG CTC AAT TTT TTT TTT TTT TTT TTT TVN- Figure 1. Visualization of the amplified cDNAs bound to the Sam68 com-
3' (V represents equal amounts of A, C and G; N represents any plex. Lanes 1-3, suppression PCR strategy. Lane 1, no template control (-
nucleotide) and the Superscript II Reverse Transcriptase (RNase H DNA). Lane 2, mock control immunoprecipitation (IP:) with normal rabbit
minus; Invitrogen Inc.) for one hour and then the beads were serum (NRS). Lane 3, anti -Sam68 immunoprecipitation with AD1 antibody.
washed with PBS:0.1% Triton X-100. The second-strand synthesis Lanes 4-6, typical double primer PCR approach (regular PCR). Lane 4, no
was performed by the addition of DNA polymerase I (New England template control. Lane 5, mock control immunoprecipitation, lane 6, anti-
Sam68 immunoprecipitation. The 'markers' indicates 1kb DNA ladder
Biolabs Inc.), E. coli RNase H (Invitrogen Inc.) and E. coli DNA (Invitrogen).
ligase (New England Biolabs Inc.) at 16˚C for two hours, as
described previously.15
Amplification of cDNAs and their identification. The cDNAs FBP11: 5'-CGG AAA GAG TCT GCC TTT AAG A-3' and 5'-
generated were amplified by linear PCR (with one primer) using TTT TCA CTG TCC CGA TCT TTT T-3', myl6: 5'-CCC CAA
0.00625 units/µl Taq DNA polymerase (Promega Inc.), 1.5 mM GAG TGA TGA GAT GAA T-3' and 5'-ATT TTA TTT GGG
MgCl2, 20 µCi dCTP α32P and 1.25 mM of the anchor primer GAA GGC AAA C-3', Ena/Vasp: 5'-CAT GGA AGA AAT GAA
(5'-GGG AGA CAA GAA TAA ACG CTC AA-3'). The conditions CAA GCT G-3' and 5'-CCA GGA GTT GAA GTT TGT TTC C-
were: 95˚C/20 s; 53˚C/20 s; 72˚C/3 min for 35 cycles. The single 3', prof1: 5'-CCA TCG TAG GCT ACA AGG ACT C-3' and 5'-
stranded DNA fragments over 500 nucleotides were gel extracted from AAT AAG GGA AAT GGG GTA ATG G-3', thrap2: 5'-GTG ACT
a denaturing 7M urea / 5% polyacrylamide gel. An RNA ligation TGA GCC AAT GTG TGA T-3' and 5'-TTC TCA CTC CTT
primer with its 3' hydroxyl group blocked with an amino modification TCT TGG CTT C-3', copeb: 5'-CGA CCA AAT TTA CCT CTG
to prevent 3' ligation (5'-PO4-CGA GAU GGC GGC UUC CUG ATC C-3' and 5'-TTA AAA GGC TTG GCA CCA GTA T-3',
C-blocked 3') was ligated at the 3' end of the amplified cDNAs with Tctp: 5'-AGG GCA AGA TGG TCA GTA GAA C-3' and 5'-ATG
T4 RNA ligase (Fermentas Inc.). Finally, the DNA fragments were CCA CCA CTC CAA ATA AAT C-3', ≤-actin: 5'-CCT GAA GTA
amplified by PCR using primers that complement the ligation and CCC CAT TGA ACA T-3' and 5'-CTG CTC GAA GTC TAG
anchor primers, but that introduce flanking EcoRI and BamHI sites AGC AAC A-3'. All oligonucleotides utilized above for the RT-PCR
for subloning in Bluescript KS to facilitate DNA sequencing at validation yield DNA fragments of 400-600 base pairs in length that
Genome Centre (McGill University). In a second strategy, a DNA were separated on 2% agarose gels and visualized by ethidium
ligation primer that complements the anchor (5'-PO4-TTG AGC bromide staining.
GTT TAT TCT TGT CTC CC-blocked 3') was utilized with T4
DNA ligase to ligate at the 3' end of the amplified cDNAs. The
DNA inserts were amplified by using suppression PCR with one
RESULTS
primer that introduces flanking EcoRI sites. Identification of mRNA targets bound to the Sam68 complex.
Validation of mRNAs associated with the Sam68 complex. To identify the mRNAs bound to endogenous Sam68, we cross-
Specific mRNAs were isolated using TRIzol (Life Technologies Inc.) linked asynchronous NIH 3T3 cells, a cell type known to express
following control or anti-Sam68 immunoprecipitations as described abundant levels of Sam68, with ultraviolet irradiation to ensure the
previously.16 Reverse transcription with the oligo dT primer was covalent association of the mRNAs with the Sam68 complex. The
performed followed by 25 to 35 cycles of PCR with the designated UV cross-linking allows the preservation of the Sam68 ribonucleo-
primers as follows: GAPDH: 5'-TGC TGA GTA TGT CGT GGA protein (RNP) complex during the immunoprecipitation. The
GTC T-3' and 5'-ATC ATA CTT GGC AGG TTT CTC C-3', bound mRNAs were converted into cDNAs by reverse transcription.

www.landesbioscience.com RNA Biology 2

 AUTHOR PLEASE PROVIDE RUNNING TITLE
Figure 2. Association of mRNAs with the endogenous Sam68 complex in
NIH 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 targets
from Table I. GAPDH served as a negative control and ≤-actin served as a
positive control. The 'markers' indicates 1kb DNA ladder (Invitrogen).
Half the eluted cDNAs were amplified by suppression PCR to favor
longer DNA fragments (Fig. 1, lanes 1–3) and the other half was
amplified by regular 2 primer PCR (Fig. 1, lanes 4–6). The DNA
fragments were purified, subcloned and sequenced (Table 1). The
Genbank accession number, the portion of the mRNA amplified,
the redundancy of each clone is shown, and whether the polyA tail
was primed are shown. Surprisingly, no intronic sequences were
found, suggesting that the captured molecules corresponded indeed
to 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 associated
with Sam68. Most likely the method was not sensitive enough or too
few clones were sequenced. The non-specific mRNAs associated
with the mock immunoprecipitations were also cloned and were
typically tRNA, ribosomal protein genes and mitochondrial gene
products (data not shown). In the clones identified with anti-Sam68
immunoprecipitations ~20% represented non-specific RNAs and
the remaining were deemed mRNAs associated with the Sam68
complex. A total of 23 mRNAs was captured with our analysis
(Table 1).
Validation of mRNA targets bound to the Sam68 complex. We
proceeded with the validation of the Sam68 captured mRNA targets.
NIH 3T3 cells were lysed and immunoprecipitated with either
control normal rabbit serum (Mock) or the anti-Sam68 antibodies.
The co-precipitating mRNAs were isolated and amplified with
primer sets corresponding to a chosen set of mRNAs from Table 1.
We used β-actin as a positive control, since it a known target of
Sam68,10,17 however we did not identify this mRNA in our assay
demonstrating that we have only identified a subset of Sam68
associated mRNAs. As a negative control gene we used the glycer-
aldehyde-3-phosphate dehydrogenase mRNA (GAPDH). It was
negative, but we increased the number of PCR cycles until we could
detect an equal amount of intrinsic noise between the mock and the
anti-Sam68 antibody immunoprecipitations. Myl6, Copeb, Fnbp3,
prof1 and Tctp were true positives and Ena/vasp and thrap2 may be
false positives as we could detect them in the corresponding mock IP
lanes (Fig. 2). These data demonstrate that ~70% of the identified
mRNAs are indeed true positives.
3 RNA Biology
DISCUSSION
We 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 were
identified as positive controls such as β-actin were not identified in
our assay. Furthermore, a correlation could not be made between the
position where reverse transcription reaction stopped, and a putative
Sam68 binding site. There was one exception: the 5' end of the
sequence for thrap2 mRNA, where the reverse transcription reaction
stopped, is a poly(U) stretch and 2 UUUA motifs. It remains to be
determined whether Sam68 binds a rather undefined site on
mRNAs, and thereby a very large set of mRNA, or whether it
requires a specific cellular context in order to bind to its RNA targets,
or any other reason thereof; one appealing theory involves mRNA
targets bound to other RNA binding proteins in the large Sam68
complex. This latter possibility explains why a specific Sam68
binding site cannot be identified with our approach.
The mRNAs identified in Table 1 can be functionally classified
into cell motility and migration (Prof1, Myl6, Evl, Fnbp3 and Bgn),
transcription-related genes (Thrap2, Copeb, Hmgn1, Hmgb1 and
Bclaf1), cell cycling/cell division (Apc5, Smc6 and Nap1l1) and cell
death (Sesn2, Anx11, Bri3bp and Bclaf1). As phosphorylation of
Sam68 by Src is known to negatively regulate RNA binding activity,18
and that Src will function to increase the motility and migration of
cancer cells, it follows that Sam68 should have a regulator effect on
its mRNA targets, or at least those related to cell motility and migra-
tion. Therefore we could speculate that tyrosine phosphorylation of
Sam68 could liberate the mRNA sequestered by Sam68, rendering
them available for translation, for example. Recent observations by
Paronetto et al., suggest that shuttling of Sam68 from the nucleus to
the cytoplasm in spermatocytes correlates with phosphorylation, that
it associates with polysomes and may facilitate translation.19
In conclusion, we have shown that specific mRNAs can be captured
to the Sam68 RNA binding protein complex. We have defined a
subset of the mRNAs that the Sam68 complex associates with
mRNAs regulating cell mobility and migration, cell cycle and tran-
scriptional regulation. Further studies are required to identify the
complete list of Sam68 RNA targets and to define the specific binding
sites in the presence or absence of certain cellular signals.
2006; Vol. 3 Issue 2
 AUTHOR PLEASE PROVIDE RUNNING TITLE

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 yes
bri3 binding protein (bri3bp) XM_132386 931–1180 3 no
non-muscle myosin light chain 3 (Myl6) U04443 114–606 3 yes
core promoter element binding protein (Copeb/KLF6/Zf9) NM_011803 998–1386 2 no
high mobility group nucleosomal binding domain 1 (Hmgn1) NM_008251 499–962 2 no
structural maintenance of chromosome 6 (Smc6) AJ310552 1368–1870 2 no
biglycan (Bgn) BC052857 1908–2375 1 yes
Ena-VASP like protein (Evl) U72519 1702–1788 1 yes
formin binding protein 3 (Fnbp3) NM_018785 1970–2348 1 no
profilin 1 (Prof1) BC002080 317–756 1 no
high mobility group box 1 BC008565 410–654 1 no
lipoprotein receptor-related protein (Lrp1) AF367720 14443–14783 1 yes
nucleosome assembly protein 1-like 1 (Nap1l1) NM_015781 1564–1795 1 no
anaphase promoting complex subunit 5 (APC5) BC046804 1917–2386 1 yes
cDNA coding for a hypothetical protein BC033455 857–1290 1 no
cDNA coding for a hypothetical protein AY061989 1781–2312 1 no
cDNA coding for a hypothetical protein NM_133718 3396–3011 1 no
sestrin 2 (Sesn2) NM_144907 2251–2533 1 no
annexin A11 (Anx11) BC012875 2040–2186 1 no
BCL2-associated transcription factor 1 (BCLAF1) BC034300 2387–2838 1 no
tumor protein, translationally-controlled 1 (Tctp) NM_009429 395–722 1 no
poly A binding protein 1 (pabc1) BC011207 2034–2214 1 yes
Chr 10, ERATO Doi 214, expressed BC021952 200–408 1 no

References
1. Lukong KE, Richard S. Sam68, the KH domain-containing superSTAR. Biochim. 14. Chen T, Boisvert FM, Bazett-Jones DP, Richard, S. A role for the GSG domain in localiz-
Biophys. Acta 2003; 1653: 73-86. ing Sam68 to novel nuclear structures in cancer cell lines. Mol Biol Cell 1999; 10:3015-33.
2. Lukong KE, Larocque D, Tyner AL, Richard S. Tyrosine phosphorylation of sam68 by 15. Gubler U, Hoffman BJ. A simple and very efficient method for generating cDNA libraries.
breast tumor kinase regulates intranuclear localization and cell cycle progression. J Biol Gene 1983; 25: 263-9.
Chem 2005; 280:38639-47. 16. Galarneau A, Richard S. Target RNA motif and target mRNAs of the Quaking STAR pro-
3. Matter N, Herrlich P, Konig H. Signal-dependent regulation of splicing via phosphoryla- tein. Nat Struct Mol Biol 2005; 12:691-8.
tion of Sam68. Nature 2002; 420: 691-5. 17. Hill MA, Schedlich L, Gunning P. Serum-induced signal transduction determines the
4. Reddy TR, Xu W, Mau JKL, Goodwin CD, Suhasini M, Tang H, Frimpong K, Rose DW, peripheral location of ≤-actin mRNA within the cell. J. Cell Biol 1994; 126: 1221-30.
Wong-Staal F. Inhibition of HIV replication by dominant negative mutants of Sam68, a 18. Wang LL, Richard S, Shaw AS. p62 association with RNA is regulated by tyrosine phos-
functional homolog of HIV-1 Rev. Nature Medicine 1999; 5:635-642. phorylation. J. Biol. Chem 1995; 270:2010-3.
5. Richard S, Torabi N, Franco GV, Tremblay GA, Chen T, Vogel G, Morel M, Cléroux P, 19. Paronetto MP, Zalfa F, Botti F, Geremia R, Bagni C, and Sette C. The nuclear RNA-bind-
Forget-Richard A, Komarova S, Tremblay ML, Li W, Li A., Gao YT, Henderson JE. ing protein Sam68 translocates to the cytoplasm and associates with the polysomes in
Ablation of the Sam68 RNA Binding Protein Protects Mice from Age-Related Bone Loss. mouse spermatocytes. Mol Biol Cell 2006; 17:14-24.
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 and
SH3 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. Mol
Cell 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 repress
cyclin D1 expression and inhibits its ability to induce apoptosis. Oncogene 2002; [Epub
ahead of print].
13. Babic I, Jakymiw A, Fujita DJ. The RNA binding protein Sam68 is acetylated in tumor cell
lines, and its acetylation correlates with enhanced RNA binding activity. Oncogene 2004;
23:3781-9.

www.landesbioscience.com RNA Biology 4