Conserved stem-loop structures in the HIV-1 RNA region containing the A3 3' splice site and its cis-regulatory element: possible involvement in RNA splicing

Abstract

The HIV-1 transcript is alternatively spliced to over 30 different mRNAs. Whether RNA secondary structure can influence HIV-1 RNA alternative splicing has not previously been examined. Here we have determined the secondary structure of the HIV-1/BRU RNA segment, containing the alternative A3, A4a, A4b, A4c and A5 3' splice sites. Site A3, required for tat mRNA production, is contained in the terminal loop of a stem-loop structure (SLS2), which is highly conserved in HIV-1 and related SIVcpz strains. The exon splicing silencer (ESS2) acting on site A3 is located in a long irregular stem-loop structure (SLS3). Two SLS3 domains were protected by nuclear components under splicing condition assays. One contains the A4c branch points and a putative SR protein binding site. The other one is adjacent to ESS2. Unexpectedly, only the 3' A residue of ESS2 was protected. The suboptimal A3 polypyrimidine tract (PPT) is base paired. Using site-directed mutagenesis and transfection of a mini-HIV-1 cDNA into HeLa cells, we found that, in a wild-type PPT context, a mutation of the A3 downstream sequence that reinforced SLS2 stability decreased site A3 utilization. This was not the case with an optimized PPT. Hence, sequence and secondary structure of the PPT may cooperate in limiting site A3 utilization.

hnRNP A1 controls HIV-1 mRNA splicing through cooperative binding to intron and exon splicing silencers in the context of a conserved secondary structure

“…Inefficient splicing of the HIV-1 genome is required to preserve a pool of unspliced and singly spliced transcripts, to be transported across the nuclear membrane via the Rev/RRE dependent pathway+ Besides having suboptimal splice sites, exonic and intronic sequences in the tat pre-mRNA function as binding sites for cellular factors of the hnRNP A/B and hnRNP H classes, which leads to further inhibition of splicing (Amendt et al+, 1994(Amendt et al+, , 1995Staffa & Cochrane, 1994, 1995Si et al+, 1997Si et al+, , 1998Caputi et al+, 1999 Our computer predictions and probing results are consistent with the existence of three major stem loop structures, SL1, SL2, and SL3, of which SL1 and SL3 are conserved in M-type HIV-1+ The ISS and the ESS3 are positioned within SL1 and SL3, respectively, whereas the SL2 harbors a GAA repeat at positions 8420-8430, which is part of the degenerate SF2/ASF-dependent enhancer element ESE3 (Mayeda et al+, 1999;Tange & Kjems, 2001)+ This GAA repeat is highly accessible to chemical modifications by DMS/DEP and single-strand specific RNase cleavage (Fig+ 2A, B), consistent with these sequences acting as binding sites for cellular splicing proteins including hnRNP A1 and presumably also SF2/ASF+ In contrast, we found that S3, which is the functionally most hnRNP A1 responsive site of the ISS, was less accessible to enzymatic probing reagents and situated in a helix structure adjacent to an internal bulge+ Intriguingly, this structure is very similar to the recently reported secondary structural context of the ESS2 (Jacquenet et al+, 2001b)+ The functional significance of this structure is supported by the observation that this base-pairing scheme can form in all investigated M-group viruses (Fig+ 3)+ Another similarity between the ISS and the ESS2 is the appearance of a base-paired UAG element immediately upstream, which corresponds to the S2 sequence of the ISS (Jacquenet et al+, 2001b; this report)+ Modulation of hnRNP A1 binding by RNA structure hnRNP A1 has originally been reported to have a general nonspecific RNA-binding activity, although several reports have specified RNA sequences that are bound by hnRNP A1 with 10-1,000-fold increased affinity (Swanson & Dreyfuss, 1988;Buvoli et al+, 1990;Matunis et al+, 1993;Burd & Dreyfuss, 1994;Mayeda et al+, 1994Mayeda et al+, , 1998Chabot et al+, 1997;Blanchette & Chabot, 1999;Caputi et al+, 1999;Del Gatto-Konczak et al+, 1999;…”

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).…”

“…PCR products were quantified using ImageQuant (Amersham Biosciences). The fractionated cDNAs were identified by reference to previous data on HeLa cells transfected with plasmid ⌬PSP (11,24).…”

“…(ii) Their branch point sequences are not canonical, and in some cases, a residue other than an adenosine is used as the branch site (22,23). (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).…”

“…The present knowledge in this field can be summarized as follows: (i) deep studies, which were performed both in vitro and in vivo, demonstrated the action of proteins ASF/SF2 and SC35 at acceptor site A7 (17,19,20,39,40); (ii) an in vitro study performed with S100 extract showed that, among the SR proteins SC35, SRp40, SRp55, and SRp70, only protein SC35 is able to activate site A3 in S100 extract (41). For a complete study of the effects of individual SR proteins on the entire set of HIV-1 splicing sites in cellular conditions, we cotransfected HeLa cells with a construct that produces a truncated HIV-1 RNA containing all the splicing sites (13,24), and a plasmid that overexpresses one of the SC35, SRp40, ASF/SF2, and 9G8 SR proteins. In these ex vivo experiments, very different HIV-1 RNA splicing patterns were obtained with different overexpressed SR protein.…”