Intronic sequences and 3'' splice sites control Rous sarcoma virus RNA splicing.

cis-acting sequences of Rous sarcoma virus (RSV) RNA involved in control of the incomplete splicing that is part of the retroviral life cycle have been studied. The 5' and two alternative 3' splice sites, as well as negative regulator of splicing element in the intron, have been introduced into chimeric constructs, and their responsive roles in splicing inhibition have been evaluated by transient transfection experiments. Although the RSV 5' splice site was used efficiently in these assays, substrates containing either the RSV env or the RSV src 3' splice site were not spliced completely, resulting in 40 to 50% unspliced RNA. Addition of the negative regulator of splicing element to substrates containing RSV 3' splice sites resulted in greater inhibition of splicing (70 to 80% unspliced RNA), suggesting that the two elements function independently and additively. Deletion of sequences more than 70 nucleotides upstream of the src 3' splice site resulted in efficient splicing at this site, suggesting that inefficient usage is not inherent in this splice site but is instead due to to sequences upstream of it. Insertion of these upstream sequences into the intron of a heterologous pre-mRNA resulted in partial inhibition of its splicing. In addition, secondary structure interactions were predicted to occur between the src 3' splice site and the inhibitory sequences upstream of it. Thus, RSV splicing control involves both intronic sequences and 3' splice sites, with different mechanisms involved in the underutilization of the env and src splice acceptor sites.     

Sex-specific alternative splicing of RNA from the transformer gene results from sequence-dependent splice site blockage

Sex-specific alternative splicing of RNA from the Drosophila transformer gene involves competition between two 3' splice sites. In the absence of Sex-lethal activity (as in males), only one site functions; in the presence of Sex-lethal activity (as in females), both sites function. Information for sex-specific splice site choice is contained within the intron itself. Deletions of the splice site used in males lead to Sex-lethal-independent use of the otherwise female-specific site. The relative amounts of unspliced and spliced RNA derived from these mutant genes do not change with changes in Sex-lethal activity. Specific nucleotide changes in the non-sex-specific splice site do not affect splicing activity but eliminate Sex-lethal-induced regulation. A deletion removing material between the two splice sites does not eliminate sex-specific regulation, while a deletion of the female splice site leads to a female-specific increase in unspliced RNA. These results are consistent with a model in which female-specific factors block the function of the non-sex-specific 3' splice site.     

Control of Human Immunodeficiency Virus Type 1 RNA Metabolism: Role of Splice Sites and Intron Sequences in Unspliced Viral RNA Subcellular Distribution

In the course of examining the various factors which affect the metabolism of human immunodeficiency virus type 1 (HIV-1) RNA, we examined the role of intron sequences and splice sites in determining the subcellular distribution of the RNA. Using in situ hybridization, we demonstrated that in the absence of Rev, unspliced RNA generated with an HIV-1 env expression construct displayed discrete localization in the nucleus, coincident with the location of the gene and not associated with SC35-containing nuclear speckles. Expression of Rev resulted in a disperse signal for the unspliced RNA throughout both the nucleus and the cytoplasm. Subsequent fractionation of the nucleus revealed that the majority of unspliced viral RNA within the nucleus is associated with the nuclear matrix and that upon expression of Rev, a small proportion of the unspliced RNA is found within the nucleoplasm. Mutations which altered splice site utilization did not alter the sequestration of unspliced RNA into discrete nuclear regions. In contrast, a 2.2-kb deletion of intron sequence resulted in a shift from discrete regions within the nucleus to a disperse signal throughout the cell, indicating that intron sequences, and not just splice sites, are required for the observed nuclear sequestration of unspliced viral RNA.