A Second Exon Splicing Silencer within Human Immunodeficiency Virus Type 1 tat Exon 2 Represses Splicing of Tat mRNA and Binds Protein hnRNP H

Abstract

An equilibrium between spliced and unspliced primary transcripts is essential for retrovirus multiplication. This equilibrium is maintained by the presence of inefficient splice sites. The A3 3-splice site of human immunodeficiency virus type I (HIV-1) is required for Tat mRNA production. The infrequent utilization of this splice site has been attributed to the presence of a suboptimal polypyrimidine tract and an exonic splicing silencer (ESS2) in tat exon 2 ϳ60 nucleotides downstream of 3-splice site A3. Here, using site-directed mutagenesis followed by analysis of splicing in vitro and in HeLa cells, we show that the 5 extremity of tat exon 2 contains a second exonic splicing silencer (ESS2p), which acts to repress splice site A3. The inhibitory property of this exonic silencer was active when inserted downstream of another HIV-1 3-splice site (A2). Protein hnRNP H binds to this inhibitory element, and two Uto-C substitutions within the ESS2p element cause a decreased hnRNP H affinity with a concomitant increase in splicing efficiency at 3-splice site A3. This suggests that hnRNP H is directly involved in splicing inhibition. We propose that hnRNP H binds to the HIV-1 ESS2p element and competes with U2AF 35 for binding to the exon sequence flanking 3-splice site A3. This binding results in the inhibition of splicing at 3-splice site A3.Because the unique transcript produced from the integrated proviral cDNA of retroviruses serves as the genome for newly synthesized virions and also for the production of mRNAs by alternative splicing, retrovirus multiplication depends upon an equilibrium between spliced and unspliced primary transcripts. To ensure this equilibrium, retroviral RNAs generally have splice sites that are used with low efficiencies. In human immunodeficiency virus type I (HIV-1), 3Ј-splice sites (3Јss) 1 and several central 3Јss (A3, A4a, A4b, A4c, and A5) compete with each other (Fig. 1A). Site A3 is required for production of tat mRNAs, sites A4a, b, and c for production of rev and env mRNAs and site A5 for production of nef and env mRNAs (Fig. 1A) (1). Metazoan 3Јss consist of three critical elements: the branchpoint sequence (2, 3), a polypyrimidine tract (PPT) sequence (4, 5) and an AG dinucleotide at the 3Ј-end of the intron (for reviews, see Refs. 6 -9). HIV-1 branchpoint sequences are highly divergent in comparison to the metazoan consensus sequence (10 -12), and HIV-1 PPTs are suboptimal (short and interspersed by purines) (13-15). The affinity of factor U2AF for the PPT depends upon the presence of a long stretch of U residues (9, 16). Factor U2AF consists of two proteins, U2AF 65 and U2AF35 , with molecular weight of 65 and 35, respectively (17). Introns with suboptimal PPTs, like those in HIV-1 RNA, require binding of U2AF 35 at the intron-exon junction for stable interaction of U2AF 65 with the PPT (9, 18, 19). Furthermore, suboptimal 3Јss are often the subject of positive or negative regulation by cis-regulatory elements, which are frequently located in the 3Ј exon. Exonic splicing en...

Mechanisms of activation and repression by the alternative splicing factors RBFOX1/2

“…hnRNP H1 belongs to the superfamily of heterogeneous nuclear ribonucleoproteins (hnRNPs). It binds to intronic oligo-(G) sequences and regulates alternative exons negatively or positively, depending upon the context (Chen et al 1999;Chou et al 1999;Jacquenet et al 2001;Caputi and Zahler 2002;Garneau et al 2005;Crawford and Patton 2006). It was recently shown that hnRNP F and hnRNP H form a complex with RBFOX2, and this interaction enhances hnRNP H/F's ability to antagonize SRSF1 binding to FGFR2 exon IIIc (Mauger et al 2008).…”

Differential Effects of the SR Proteins 9G8, SC35, ASF/SF2, and SRp40 on the Utilization of the A1 to A5 Splicing Sites of HIV-1 RNA

“…Plasmids Used in This Study-pLD-C2, pLD-C3, and pLD-L3-U1 constructs, used for production of the C2, C3, and L3-U1 transcripts, were described previously (11,23,24). To build the plasmid pLD-C1, two DNA fragments were generated by PCR amplifications of plasmid pBRU3 (42) containing the HIV-1 BRU/LAI complete cDNA (GenBank TM accession number K02013).…”

“…The combination of these various sites gives rise to at least 35 different mRNAs (1). Although the relative efficiencies of the HIV-1 donor sites seem to depend mainly upon their complementarity to the U1 snRNA 5Ј-terminal sequence (3,4), efficiencies of HIV-1 acceptor sites depend upon the presence of cis-regulatory elements (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). Several studies have shown that HIV-1 acceptor sites are suboptimal as follows.…”

“…(iii) Their accessibility is limited by their sequestering in stable secondary structures (24). (iv) Several cis-inhibitory elements have been identified that down-regulate the A2, A3, and A7 sites by binding of hnRNP A1 or hnRNP H proteins (11,12,14,16,18,19,21). Despite these numerous handicaps, the utilization of several of the HIV-1 acceptor sites is essential for virus multiplication.…”

Structural basis of G-tract recognition and encaging by hnRNP F quasi-RRMs

“…Some of the trans-acting factors that bind to these G-tracts belong to the heterogeneous nuclear ribonucleoprotein (hnRNP) F/H family of proteins that consists of five members (hnRNP F, hnRNP H, hnRNP H', hnRNP 2H9, and GRich Sequence Factor (GRSF) 1) 9,10 . GRSF-1 is mainly involved in translation regulation 11,12 and Internal Ribosome Entry Site (IRES) mediated translation 13 , while hnRNP H, H' and F are mostly involved in the regulation of alternative splicing [14][15][16][17][18][19][20][21][22][23] and polyadenylation [24][25][26] . The posttranscriptional regulation by hnRNP F/H proteins is a crucial event since mutations in G-tracts often result in aberrant splicing patterns and can be associated with various diseases [27][28][29][30][31][32] .…”