In vitro splicing of mutually exclusive exons from the chicken beta-tropomyosin gene: role of the branch point location and very long pyrimidine stretch.

The E1A pre-mRNA of adenovirus is spliced into three mRNA species (13S, 12S, and 9S mRNAsl by the use of three alternative 5'-splice sites. The 13S and 9S mRNAs predominate during the early and late periods of infection, respectively. With HeLa nuclear extracts isolated in early and late periods of infection, we were able to reproduce a 13S-9S modulation that resembles that occurring in infected cells. An in vitro analysis of the c/s-acting parameters involved in the 13S-9S switch indicates that the 13S mRNA splicing inhibition is one of the first events of the late period and leads to the subsequent stimulation of the 9S mRNA reaction. The new abilities of the late nuclear extract for the 9S mRNA reaction were also confirmed by analyzing splicing of a major late transcript containing leaders 1 and 2 separated by the wild-type intervening sequence (IVS) of 1021 nucleotides. Complementation experiments show that the trans-acting factor(sl are micrococcal nuclease sensitive. They were partially characterized by induction experiments, and we show that the primary factors responsible for the 13S-9S modulation in vitro are viral RNAs of high molecular weight that accumulate late in infection. We postulate that the splicing modulation of E1A pre-mRNA results from an indirect mode of action for these viral RNAs, based on a sequestration of common splicing factors that are not present in vast excess in HeLa cells.

Section: Discussionmentioning

confidence: 85%

Modulation of alternative splicing of adenoviral E1A transcripts: factors involved in the early-to-late transition.

Gattoni¹,

Chébli²,

Himmelspach³

et al. 1991

“…The molecular basis for this is the proximity of the 5'-splice site of exon 2 relative to the branchpoint used for exon 3, which is only 42 nucleotides apart. In contrast, this mechanism cannot explain the mutually exclusive use of exons 6 and 7 in the rat and chicken B-TM genes, because the 5'-splice site of exon 6 in these genes is located at least 136 nucleotides upstream of the branchpoints used for the skeletal muscle exon (Helfman et al 1989(Helfman et al , 1990Goux-Pelletan et al 1990). Thus, the distant branchpoints of the [3-TM genes in rat and chicken play a different role in alternative splicing.…”

Section: The Role Of Distant Branch Sites In Alternative Splice-site mentioning

confidence: 96%

“…cellular factors involved in regulated alternative splicing, but a number of features in the pre-mRNA have been implicated in alternative splice-site selection. These include the relative strengths of 5'-and 3'-splice sites (Zhuang et al 1987;Kuo et al 1991;Mullen et al 1991), intron size (Fu and Manley 1987), the pyrimidine content of a 3'-splice site (Fu et al 1988;Mullen et al 1991), the location of branchpoints (Gattoni et al 1988;Helfman and Ricci 1989;Goux-Pelletan et al 1990;Helfman et al 1990), multiple alternative branchpoints (Noble et al 1987(Noble et al , 1988Gattoni et al 1988;Helfman and Ricci 1989), branchpoint sequences (Reed and Maniatis 1988;Zhuang et al 1989;Mullen et al 1991 ), intron sequences between a 3'-splice site and upstream branchpoint Helfman et al 1990;Libri et al 1990), and exon sequences (Reed and Maniatis 1986;Mardon et al 1987;Somaseker and Mertz 1985;Helfman et al 1988;Cooper and Ordahl 1989;Hampson et al 1989;Streuli and Saito 1989;Black 1991;Libri et al 1990Libri et al , 1991.…”

mentioning

confidence: 99%

Alternative splicing of beta-tropomyosin pre-mRNA: cis-acting elements and cellular factors that block the use of a skeletal muscle exon in nonmuscle cells.

Guo¹,

Mulligan²,

Wormsley³

et al. 1991

100

The rat I~-tropomyosin (13-TM) gene encodes both skeletal muscle 13-TM and fibroblast TM-1 by an alternative RNA-splicing mechanism. This gene contains 11 exons. Exons 1-5, 8, and 9 are common to all mRNAs expressed from the gene. Exons 6 and 11 are used in fibroblasts as well as smooth muscle cells, whereas exons 7 and 10 are used in skeletal muscle cells. In this study we have carried out an extensive mutational analysis to identify c/s-acting elements that block the use of the skeletal muscle-specific exon 7 in nonmuscle cells. These studies localize the critical elements for regulated alternative splicing to sequences within exon 7 and the adjacent upstream intron. In addition, mutations that inactivate the 5'-or 3'-splice sites of exon 6 do not result in the use of the skeletal muscle-specific exon 7 in nonmuscle cells, suggesting that splice-site selection in vivo is not regulated by a simple c/s-acting competition mechanism but, rather, by a mechanism that inhibits the use of exon 7 in certain cellular environments. In support of this hypothesis we have identified sequence-specific RNA-binding proteins in HeLa cell nuclear extracts using native gel electrophoresis and binding competition assays. Mutations in the pre-mRNA that result in the use of the skeletal muscle exon in vivo also disrupt the binding of these proteins to the RNA in vitro. We propose that the binding of these proteins to the pre-mRNA is involved in regulated alternative splicing and that this interaction is required for blocking the use of the skeletal muscle exon in nonmuscle cells.

“…These results were confirmed by primer extension analysis of branched and debranched RNA substrates (data not shown). Although functional BPSs are usually located within 18-40 nucleotides upstream of the AG, there are cases in which lariat formation occurs farther upstream (Gattoni et al 1988;Helfman and Ricci 1989;Reed 1989;Goux-Pelletan et al 1990). In these cases, the branchpoint sequence is followed immediately by a pyrimidine stretch.…”

Section: Productive and Nonproductive Branchpointmentioning

confidence: 99%

The role of branchpoint and 3'-exon sequences in the control of balanced splicing of avian retrovirus RNA.

Fu¹,

Katz²,

Skalka³

et al. 1991

110

128

We previously described an avian sarcoma-leukosis virus (ASLV) insertion mutation that causes a decrease in the ratio of unspliced to spliced RNA in vivo, resulting in a replication defect. Pseudorevertant viruses containing cis-acting suppressor mutations that restored the normal ratio were isolated. One class of the suppressor mutations consists of single-base changes or small deletions near the 3' splice site, while another consists of deletions in the 3' exon. In this paper we report results from an in vitro analysis of wild-type, mutant, and pseudorevertant pre-mRNA splicing. We find that wild-type RNA is spliced inefficiently in vitro, and that the insertion mutation and suppressors act directly at the level of splicing. Characterization of splicing intermediates reveals that the insertion mutation and suppressor mutations located within the intron alter the pattern of lariat formation. In contrast, suppressor mutations consisting of 3' exon deletions act at an earlier step in the splicing pathway. Thus, the efficiency of splicing at the env 3' splice site can be affected at the level of spliceosome assembly, lariat formation, or cleavage at the 3' splice site and exon ligation.[Key Words: Retrovirus; splicing; splice site selection] Received September 18, 1990; revised version accepted December 13, 1990.Retrovirus RNA transcripts are subject to multiple pathways of RNA processing and transport (for a review, see Stoltzfus 1989). The primary full-length RNA po7lI transcript of -7-10 kb functions as genomic RNA for progeny virions and mRNA for structural proteins and enzymes. Both of these functions require transport of an intact viral RNA to the cytoplasm. In addition, a fraction of the full-length transcripts is spliced in the nucleus to generate subgenomic mRNAs that encode other viral proteins (Fig. lA). Thus, a balance between full-length RNA and spliced viral mRNAs is required for replication of retroviruses.Multiple CIS-acting sequences have been shown to play a role in splice site selection in cellular pre-mRNAs (for review, see Krainer and Maniatis 1988). These sequences include the 5' and 3' splice sites, the branchpoint sequence (BPS), and sequences within the flanking exons. Interaction of these cis-acting elements with small nuclear ribonuclear proteins (snRNPs) and many protein splicing factors leads to the assembly of spliceosomes in which the splicing reaction takes place (see Krainer and Maniatis 1988;Steitz et al. 1988; for recent reviews, see Bindereif and Green 1990).Retroviral transcripts are presumably processed by the same mechanisms as cellular pre-mRNAs. However, unlike most cellular pre-mRNAs, a portion of viral RNA transcripts escape splicing entirely. Balanced splicing of retroviral RNA is therefore a novel form of alternative splicing. An understanding of the mechanisms involved in this splicing may reveal both general and novel features of the splice site selection process. Several models for maintaining the balance of spliced and unspliced forms of retrovirus RNA have been pro...