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.Generation of mature mRNA in eukaryotes generally requires multiple processing steps, including capping, splicing, and polyadenylation, that are coupled to ensure proper processing (reviewed in reference 31). Retroviruses utilize the host transcription/RNA processing machinery to generate viral RNA, but due to peculiarities of their replication scheme, they often utilize the RNA processing machinery in unique ways. In the simple avian retrovirus Rous sarcoma virus (RSV), the env and src mRNAs are generated by RNA splicing from a common 5Ј splice site (ss) to one of two alternative 3Ј ss (10). However, unlike most host genes, retrovirus replication requires that a substantial portion of the primary viral transcripts remain completely unspliced to serve as gag-pol mRNA and as genomic RNA for progeny virions. RSV employs several mechanisms to preserve the pool of unspliced RNA, including the maintenance of suboptimal 3Ј ss (25, 59) and the action of splicing repressor elements within the gag gene (the negative regulator of splicing, or NRS) (3, 39, 49) and upstream of the src 3Ј ss (the suppressor of src splicing) (1, 39, 50). Generation of functional unspliced viral mRNA poses problems for the coupling of the splicing and polyadenylation reactions.The NRS has been well characterized and is thought to act as a pseudo-5Ј ss that sequesters viral 3Ј ss in a nonproductive splicing complex (reviewed in reference 9). An upstream region of the ϳ230-nucleotide (nt) element bind...

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.

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.