The Exon Splicing Silencer in Human Immunodeficiency Virus Type 1 Tat Exon 3 Is Bipartite and Acts Early in Spliceosome Assembly

Abstract

Inefficient splicing of human immunodeficiency virus type 1 (HIV-1) RNA is necessary to preserve unspliced and singly spliced viral RNAs for transport to the cytoplasm by the Rev-dependent pathway. Signals within the HIV-1 genome that control the rate of splicing include weak 3 splice sites, exon splicing enhancers (ESE), and exon splicing silencers (ESS). We have previously shown that an ESS present within tat exon 2 (ESS2) and a suboptimal 3 splice site together act to inhibit splicing at the 3 splice site flanking tat exon 2. This occurs at an early step in spliceosome assembly. Splicing at the 3 splice site flanking tat exon 3 is regulated by a bipartite element composed of an ESE and an ESS (ESS3). Here we show that ESS3 is composed of two smaller elements (AGAUCC and UUAG) that can inhibit splicing independently. We also show that ESS3 is more active in the context of a heterologous suboptimal splice site than of an optimal 3 splice site. ESS3 inhibits splicing by blocking the formation of a functional spliceosome at an early step, since A complexes are not detected in the presence of ESS3. Competitor RNAs containing either ESS2 or ESS3 relieve inhibition of splicing of substrates containing ESS3 or ESS2. This suggests that a common cellular factor(s) may be required for the inhibition of tat mRNA splicing mediated by ESS2 and ESS3.Human immunodeficiency virus type 1 (HIV-1) is a complex retrovirus whose RNA splicing is dependent on the host cell splicing machinery (12, 50). HIV-1 pre-mRNA contains five 5Ј splice sites and nine 3Ј splice sites which are used to generate more than 30 different mRNA species by alternative splicing. In addition, a pool of unspliced RNA must be maintained to serve as genomic RNA and as mRNA for the gag and pol gene products (18,21,32,34,35,38). To achieve the balance between spliced and unspliced RNAs, splicing of HIV-1 RNA is inefficient, a feature which is shared with all other retroviruses (4,7,11,20,22,(45)(46)(47)52). In contrast to the major HIV-1 5Ј splice sites, which are strong, the 3Ј splice sites are weak (33). These are characterized by the presence of nonconsensus polypyrimidine tracts and branch point sites other than the eukaryotic consensus adenosine residue (2,13,17,33,43). In addition, exon sequences downstream of the 3Ј splice sites enhance or inhibit splicing at the 3Ј splice sites (2,3,39,42,51). Furthermore, the virus-encoded protein Rev plays an essential role in facilitating the transport of unspliced and singly spliced mRNAs, thus preventing the viral RNA from splicing to completion (19, 37; for a review, see reference 12).HIV-1 tat mRNA is formed by the splicing of two coding exons (exon 2 and exon 3) and one or more upstream noncoding exons. Exon 3 is also joined to rev exon 2 and is therefore referred to as tat/rev exon 3 (34). The 3Ј splice sites flanking exon 2 and exon 3 (3Ј splice sites 3 and 7, respectively [ Fig. 1]) contain suboptimal polypyrimidine tracts, and the 3Ј splice site flanking exon 3 has been reported to contain a nonconsensus bra...

Determinants of 4-Repeat Tau Expression

“…This prediction was based on previous work (21) showing that this core sequence inhibits splicing of HIV tat exon 3. However, deletions E⌬9 and E⌬10 that span the TTAG sequence, rather than increasing E10 inclusion, do not alter or actually reduce E10 inclusion, respectively (Fig.…”

“…This deletion removes a previously identified cryptic exon (20). Note that the portion of HIV tat exon 3 used in pSPL3 does not contain the splicing inhibitory sequence TTAG (21) that is also present in tau E10. A 177-bp genomic fragment containing human tau E10 with 33 and 51 bp of flanking intron sequences was amplified from P1 artificial chromosome clone 4139 by PCR using forward and reverse primers I9X2 (5Ј-CCACTCGAGCGTGTCACT-CATCCTTTTTC-3Ј) and I10B2 (5Ј-CGGGATCCTAATAATTCAAGCCA-CAG-3Ј) that contain an XhoI and BamHI site, respectively.…”

“…This mutation alters the sequence TTAG, which acts as an ESS sequence in HIV-1 tat exon 3 (21). These deletions were expected to increase splicing due to deletion of the putative TTAG silencer.…”

A novel function for Sam68: Enhancement of HIV-1 RNA 3′ end processing

“…This regulation is exercised at several levels. At the level of cis-acting signals, splicing is controlled by the suboptimal nature of the 3Ј splice sites (Staffa and Cochrane 1994;O'Reilly et al 1995) as well as the exon splicing enhancers (ESEs) and exon splicing silencers (ESSs) throughout the RNA, which act to promote and inhibit recognition of the adjacent splice site, respectively (Amendt et al 1994;Staffa and Cochrane 1995;Si et al 1998;Caputi et al 1999;Bilodeau et al 2001). Moreover, the ESE and ESS within the terminal exon also regulate viral RNA transport.…”

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

“…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.…”