Characterization of Rous sarcoma virus polyadenylation site use in vitro.

Polyadenylation of Rous sarcoma virus (RSV) RNA is inefficient, as approximately 15% of RSV RNAs represent read-through transcripts that use a downstream cellular polyadenylation site (poly(A) site). Read-through transcription has implications for the virus and the host since it is associated with oncogene capture and tumor induction. To explore the basis of inefficient RSV RNA 3'-end formation, we characterized RSV polyadenylation in vitro using HeLa cell nuclear extracts and HEK293 whole cell extracts. RSV polyadenylation substrates composed of the natural 3' end of viral RNA and various lengths of upstream sequence showed little or no polyadenylation, indicating that the RSV poly(A) site is suboptimal. Efficiently used poly(A) sites often have identifiable upstream and downstream elements (USEs and DSEs) in close proximity to the conserved AAUAAA signal. The sequences upstream and downstream of the RSV poly(A) site deviate from those found in efficiently used poly(A) sites, which may explain inefficient RSV polyadenylation. To assess the quality of the RSV USEs and DSEs, the well-characterized SV40 late USEs and/or DSEs were substituted for the RSV elements and vice versa, which showed that the USEs and DSEs from RSV are suboptimal but functional. CstF interacted poorly with the RSV polyadenylation substrate, and the inactivity of the RSV poly(A) site was at least in part due to poor CstF binding since tethering CstF to the RSV substrate activated polyadenylation. Our data are consistent with poor polyadenylation factor binding sites in both the USE and DSE as the basis for inefficient use of the RSV poly(A) site and point to the importance of additional elements within RSV RNA in promoting 3' end formation.

Serine/arginine-rich proteins contribute to negative regulator of splicing element-stimulated polyadenylation in rous sarcoma virus.

Rous sarcoma virus (RSV) requires large amounts of unspliced RNA for replication. Splicing and polyadenylation are coupled in the cells they infect, which raises the question of how viral RNA is efficiently polyadenylated in the absence of splicing. Optimal RSV polyadenylation requires a far-upstream splicing control element, the negative regulator of splicing (NRS), that binds SR proteins and U1/U11 snRNPs and functions as a pseudo-5' splice site that interacts with and sequesters 3' splice sites. We investigated a link between NRS-mediated splicing inhibition and efficient polyadenylation. In vitro, the NRS alone activated a model RSV polyadenylation substrate, and while the effect did not require the snRNP-binding sites or a downstream 3' splice site, SR proteins were sufficient to stimulate polyadenylation. Consistent with this, SELEX-binding sites for the SR proteins ASF/SF2, 9G8, and SRp20 were able to stimulate polyadenylation when placed upstream of the RSV poly(A) site. In vivo, however, the SELEX sites improved polyadenylation in proviral clones only when the NRS-3' splice site complex could form. Deletions that positioned the SR protein-binding sites closer to the poly(A) site eliminated the requirement for the NRS-3' splice site interaction. This indicates a novel role for SR proteins in promoting RSV polyadenylation in the context of the NRS-3' splice site complex, which is thought to bridge the long distance between the NRS and poly(A) site. The results further suggest a more general role for SR proteins in polyadenylation of cellular mRNAs.

Analysis of RNA splicing defects in PITX2 mutants supports a gene dosage model of Axenfeld-Rieger syndrome.

BACKGROUND: Axenfeld-Rieger syndrome (ARS) is associated with mutations in the PITX2 gene that encodes a homeobox transcription factor. Several intronic PITX2 mutations have been reported in Axenfeld-Rieger patients but their effects on gene expression have not been tested. METHODS: We present two new families with recurrent PITX2 intronic mutations and use PITX2c minigenes and transfected cells to address the hypothesis that intronic mutations effect RNA splicing. Three PITX2 mutations have been analyzed: a G>T mutation within the AG 3' splice site (ss) junction associated with exon 4 (IVS4-1G>T), a G>C mutation at position +5 of the 5' (ss) of exon 4 (IVS4+5G>C), and a previously reported A>G substitution at position -11 of 3'ss of exon 5 (IVS5-11A>G). RESULTS: Mutation IVS4+5G>C showed 71% retention of the intron between exons 4 and 5, and poorly expressed protein. Wild-type protein levels were proportionally expressed from correctly spliced mRNA. The G>T mutation within the exon 4 AG 3'ss junction shifted splicing exclusively to a new AG and resulted in a severely truncated, poorly expressed protein. Finally, the A>G substitution at position -11 of the 3'ss of exon 5 shifted splicing exclusively to a newly created upstream AG and resulted in generation of a protein with a truncated homeodomain. CONCLUSION: This is the first direct evidence to support aberrant RNA splicing as the mechanism underlying the disorder in some patients and suggests that the magnitude of the splicing defect may contribute to the variability of ARS phenotypes, in support of a gene dosage model of Axenfeld-Rieger syndrome.