Shared Protein Components of SINE RNPs

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Abstract

The heterogeneous, short RNAs produced from the high, copy, short mobile elements (SINEs) interact with proteins to form RNA–protein (RNP) complexes. In particular, the BC1 RNA, which is transcribed to high levels specifically in brain and testis from one locus of the ID SINE family, exists as a discrete RNP complex. We expressed a series of altered BC1, and other SINE-related RNAs, in several cell lines and tested for the mobility of the resulting RNP complexes in a native PAGE assay to determine which portions of these SINE RNAs contribute to protein binding. When different SINE RNAs were substituted for the BC1 ID sequence, the resulting RNPs exhibited the same mobility as BC1. This indicates that the protein(s) binding to the ID portion of BC1 is not sequence specific and may be more dependent upon the secondary structure of the RNA. It also suggests that all SINE RNAs may bind a similar set of cellular proteins. Deletion of the A-rich region of BC1 RNA has a marked effect on the mobility of the RNP. Rodent cell lines exhibit a slightly different mobility for this shifted complex when compared to human cell lines, reflecting evolutionary differences in one or more of the protein components. On the basis of mobility change observed in RNP complexes when the A-rich region is removed, we decided to examine poly(A) binding protein (PABP) as a candidate member of the RNP. An antibody against the C terminus of PABP is able to immunoprecipitate BC1 RNA, confirming PABP's presence in the BC1 RNP. Given the ubiquitous role of poly(A) regions in the retrotransposition process, these data suggest that PABP may contribute to the SINE retrotransposition process.

Introduction

Short interspersed elements (SINEs) belong to a class of DNA repetitive mobile elements that undergo amplification through a retroposition process. SINEs are transcribed by RNA polymerase III (pol III), followed by the reverse transcription of the RNA, and subsequent integration of the cDNA into a new site in the genome.1., 2., 3. SINEs are set apart from other retroelements in that they do not have any protein coding capacity, and yet they have amplified to extremely high copy numbers. Because of their lack of coding capacity, they are considered non-autonomous elements, which probably rely on activities from the L1, or other, retrotransposons. Copy numbers of SINEs range anywhere from tens of thousands of copies to over a million in the case of the human SINE, Alu. However, only a very limited number of the elements within a SINE family, termed master or source elements, appear to be capable of actively undergoing retroposition.4., 5., 6. Although a variety of factors influence the retroposition process,7 details of what makes some SINEs retropositionally active are still poorly understood. Because SINEs do not code for any proteins, it has been proposed that SINEs depend on the gene products from another group of mobile elements, termed the long interspersed elements (LINEs), for the retrotransposition process. It also seems likely that both SINEs and LINEs depend on endogenous cellular proteins for some aspects of the retrotransposition process. For instance, most cellular RNAs, including SINEs8., 9. are associated with cellular proteins to form ribonucleoprotein complexes (RNPs). It is likely that the proteins in the SINE RNP influence the retrotransposition process.

The brain cytoplasmic 1 (BC1) RNA gene belongs to the rodent-specific family of SINEs, termed ID.10 Like other SINEs, it is transcribed by RNA pol III.11 BC1 is an unusual pol III transcript because it has both developmental and tissue-specific regulation being expressed only in brain and to a lesser extent in testis.12., 13., 14. Although, there are 200–120,000 copies of ID elements in a given rodent genome, only one ID element locus, the BC1 RNA gene, contributes to the very high level of neuronal expression.15 While other ID-containing loci make low levels of RNAs with heterogeneous 3′ ends,15 the BC1 locus makes large quantities of a discrete RNA species. It is likely that the upstream sequences of the BC1 locus influence its extremely high level of transcription relative to the other ID copies.14., 16., 17. The BC1 locus is the only SINE locus to date that has been demonstrated to serve as a “master” locus for SINE amplification.15 This fact along with the other unique properties of the BC1 transcript prompted us to use it as a model system to study portions of SINE RNAs and their interactions with cellular proteins. While the upstream region of BC1 contributes significantly to regulation and tissue specificity, other data indicate that post-transcriptional regulation also plays a role.17 One possible post-transcriptional variable may be the proteins that bind to BC1 RNA to form the endogenous RNP.9., 10. The RNP has been shown to form in all rodent tissues when BC1 is artificially expressed in transgenic animals. In addition, the RNP is able to form at the earliest developmental time points at which BC1 RNA is expressed.17 These data indicate that BC1 RNP proteins should be abundant and ubiquitously expressed. There may be different proteins involved at different stages of BC1 RNA expression, with some involved in nuclear stability and transport, as well as others involved in the cytoplasmic complex. BC1 must be transiently present in the nucleus, but because most of the mass of BC1 RNA exists as a cytoplasmic RNP, our studies address only this final complex.

BC1 RNA consists of three sequence domains.16 The first 75 nucleotides is the portion derived from tRNAAla that gives rise to ID elements18 and forms a fairly stable hairpin structure.19 An A-rich region (53 bases in BC1) is also present in all SINE transcripts, but varies in length (from zero to greater than 100 bases) and in A content. This A-rich region is the one feature that is common to almost all of the non-LTR retrotransposons, and is thought to play a critical role in the priming of reverse transcription.20 The unique portion of the 3′ end is different for each SINE transcript and is therefore the only portion of the transcript that varies greatly between loci. The 3′ end of BC1 RNA is a unique sequence which has been highly conserved at that locus throughout rodent evolution.16

The proteins that comprise the BC1 RNP are still unknown, although several candidates have been proposed.21., 22., 23. Studies have characterized the nuclear complex,22., 24. or have used in vitro assays with co-purification correlations23., 25. to suggest candidate BC1 RNP components. No direct manipulation of BC1 RNA itself has been tested for impact on the cytoplasmic RNP formation. Here we have created constructs expressing transcripts where one or more of the three sequence domains have been removed or altered. We then characterized the cytoplasmic RNP complexes made with these various altered SINE RNAs to determine which sequence(s) is important for the formation of the endogenous BC1 RNP complex.

Section snippets

Native mobility shift assay and construct design

Our approach to studying the RNPs formed with BC1 and other SINEs was to perform electrophoretic mobility shift analysis by neutral PAGE using crude cytoplasmic extracts to look at the endogenous RNPs. In order to study mutant or altered RNA structures, we used transfection experiments in cultured cells to introduce plasmids that express variant RNAs into the cells. Although in some situations the complexes may change after cell lysis, we believe that this assay is best for detecting authentic

Discussion

SINE elements are small retroposons present within all eukaryotes. All SINEs are ancestrally derived from either the 7SL RNA gene or tRNA genes. Primate and rodent SINEs are the most intensely studied and most abundant of the SINE elements. The function of SINEs, if any, is unproven but their effects are well documented.27., 28. SINEs affect the host genome in a number of ways. By replicating and re-inserting into the genome, the site in which they land can be dramatically altered and its

Construction of plasmids

The variants of the p7SLBC1BC1 construct17 were derived by PCR amplification using primers that incorporated the specific nucleotide changes desired. Nomenclature of the vectors follows our previously described format17 where the upstream region is superscripted, followed by the SINE body, and then the unique 3′ end of the transcript (including the terminator) is again in superscript. The final PCR product of the complete construct was cloned using the pGEM-Teasy vector system kit (Promega)

Acknowledgements

A.M.R. was supported by a Brown Foundation fellowship from the Tulane Cancer Center. This research was supported by National Institutes of Health RO1 GM45668 and by the Department of the Army DAMD17-98-1-8119 (to P.L.D.). We thank Dr N. Hecht at the University of Pennsylvania Medical Center for generously supplying the anti-TB-RBP antibody and Dr J. Keene at Duke University Medical Center, Durham, North Carolina for kindly supplying the several anti-La antisera used in this study.

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