(23,41). In fact, the presence of an intron can influence the way in which at least one nuclear factor associates with the RNA sequences that direct polyadenylation. This was evident with the demonstration that crosslinking of the heterogeneous nuclear ribonucleoprotein C polypeptide to RNA sequences within the SV40 late polyadenylation site is altered by the insertion of an intron, although the functional significance of the alteration is not understood (46). Conversely, mutation of the AAUAAA polyadenylation signal has been shown by using nuclear extracts to depress splicing of the final intron but not the penultimate intron (37). Somewhat at odds with the data that functionally link the final intron to the process of 3'-end formation was the finding that the same chimeric RNA that was used in the Niwa et al. (39) study could be cleaved and polyadenylated after it had been spliced, provided that it was first extracted from agarose and deproteinized (36). The kinetics of cleavage and polyadenylation were similar to those for the unspliced species and, therefore, contraindicated a role of the final intron in 3'-end formation. We rationalized that studies using intact cells so that all cellular factors would be present at physiological concentrations should help to resolve the function of the final intron in 3'-end formation. If a function were indicated, then it would also be possible to localize the relevant intron sequences by deletion analysis.In this report, we begin to examine in cultured cells the 3359 Vol. 13,No. 6 on April 30, 2019 by guest
For all intron-containing pre-mRNAs of higher eukaryotes that have been examined using either living cells or cell-free extracts, a functional 3' splice site within the 3'-terminal intron is required for efficient RNA 3'-end formation. The mechanism by which intron sequences facilitate RNA 3'-end formation, which is achieved by endonucleolytic cleavage and polyadenylation, is not understood. We report here that in intact cells the efficiency of RNA 3'-end formation correlates with the efficiency of final intron removal, even when the intron is normally a 5'-terminal or internal intron. Therefore, the influence of the 3'-terminal intron on 3'-end formation is likely to be attributable to the determinants of splicing efficiency, which include but are not limited to the 3' splice site. Quantitative RNase mapping and methods that couple reverse transcription and the polymerase chain reaction were used to assess the consequence to RNA 3'-end formation of intron deletions within the human gene for triosephosphate isomerase (TPI). Results indicate that the formation of TPI RNA 3' ends requires TPI gene introns in addition to the last intron, intron 6, to proceed efficiently. These additional TPI gene introns are also required for the efficient removal of intron 6. When introns 1 and 5 were engineered to be the final intron, they were found, as was intron 6, to function in RNA 3'-end formation with an efficiency that correlated with their efficiency of removal. The simultaneous deletion of the 5' and 3' splice sites of intron 6 reduced the efficiencies of both RNA 3'-end formation and the removal of intron 5, which constituted the 3'-most functional intron. Deletion of only the 3' splice site of intron 6 precluded RNA 3'-end formation but had no effect on the efficiency of intron 5 removal. Deletion of only the 5' splice site of intron 6, which resulted in exon 6 skipping (i.e., the removal of intron 5, exon 6, and intron 6 as a single unit), had no effect on the efficiencies of either RNA 3'-end formation or the removal of intron 5-exon 6-intron 6. These results indicate that sequences within the 3'-terminal intron are functionally coupled to both RNA 3'-end formation and removal of the penultimate intron via a network of interactions that form across the last two exons and, most likely, between RNA processing factors.[Key Words: Pre-mRNA processing; polyadenylation; splicing) Received September 7, 1993; revised version accepted December 17, 1993.Most pre-mRNAs of higher eukaryotes are processed in the nucleus by a series of steps that include capping at the 5' end, intron removal by splicing, and polyadenylation at the 3' end. The pre-mRNA sequences and transacting factors that are required for splicing (Green 1991;Guthrie 1991; Wassarman and Steitz 1992) and polyadenylation (Wahle and Keller 1992) have been studied extensively. Splicing is directed by conserved sequences at the 5' and 3' ends of introns that interact either directly or indirectly with small nuclear ribonucleoproteins (snRNPs), including Ul, U2, U5, ...
Evidence exists from studies using intact cells that intron removal can be influenced by the reactivity of upstream and downstream splice sites and that cleavage and polyadenylation can be influenced by the reactivity of upstream splice sites. These results indicate that sequences within 3-terminal introns can function in the removal of upstream introns as well as the formation of RNA 3 ends. Evidence from studies using intact cells for an influence of RNA 3-end formation on intron removal is lacking. We report here that mutations within polyadenylation sequences that either decrease or increase the efficiency of RNA 3-end formation have no effect on the efficiencies with which either the 3-terminal or the penultimate intron is removed by splicing. Northern (RNA) blot hybridization, RNase mapping, and an assay that couples reverse transcription and PCR were used to analyze the effects of deletions and a substitution of the polyadenylation sequences within the human gene for triosephosphate isomerase (TPI). TPI pre-mRNA harbors six introns that are constitutively removed by splicing. Relative to normal levels, each of the deletions was found to reduce the nuclear and cytoplasmic levels of TPI mRNA, increase the nuclear level of unprocessed RNA 3 ends, and decrease the nuclear level of processed RNA 3 ends. In contrast, the substitution using the polyadenylation sequences of the mouse  major -globin gene was found to increase the nuclear and cytoplasmic levels of TPI mRNA, decrease the nuclear level of unprocessed 3 ends, and increase the nuclear level of processed 3 ends. The simplest interpretation of these data indicates that (i) the rate of 3-end formation normally limits the amount of mRNA produced and (ii) the deletions decrease and the substitution increases the efficiency of RNA 3-end formation. While each of the deletions and the substitution altered the absolute levels of intron 6-containing, intron 5-containing, intron 6-free, and intron 5-free RNAs, in no case was there an abnormal ratio of intron-containing to intron-free RNA for either intron. Therefore, at least for TPI RNA, while the efficiency of removal of the 3-terminal intron influences the efficiency of RNA 3-end formation, the efficiency of RNA 3-end formation does not influence the efficiency of removal of either the 3-terminal or penultimate intron. The dependence of TPI RNA 3-end formation on splicing may reflect the suboptimal strengths of the corresponding regulatory sequences and may function to ensure that TPI pre-mRNA is not released from the chromatin template until it has formed a complex with spliceosomes. If so, then the independence of TPI RNA splicing on 3-end formation may be rationalized by the lack of a comparable function.Most vertebrate pre-mRNAs are processed via steps in the nucleus that include capping at the 5Ј end (39), polyadenylation at the 3Ј end (43), and splicing to remove introns (11,13,31,44). The mechanism of polyadenylation, which involves an endonucleolytic cleavage and the subsequent addition of 50 to 250 a...
In cultured cells, little if any mRNA accumulates from an intronless version of the human gene for triosephosphate isomerase (TPI), a gene that normally contains six introns. By deleting introns either individually or in combinations, it was demonstrated by Northern (RNA) blot hybridization that while the deletion of a greater number of introns generally results in a lower level of product mRNA, not all introns contribute equally to mRNA formation. For example, intron 1 appeared to be dispensable, at least when the remaining introns are present, but deletion of the last intron, intron 6, reduced the level of product mRNA to 51% of normal. To determine how intron 6 contributes to mRNA formation, partial deletions of intron 6 were constructed and analyzed. Deletion of the lariat and acceptor splice sites or the donor, lariat, and acceptor splice sites, each of which precluded removal of the intron 6 sequences that remained, reduced the level of product mRNA to < 1 or 27% of normal, respectively. As measured by RNase mapping and cDNA sequencing, the decrease in mRNA abundance that was attributable to the complete and partial intron 6 deletions was accompanied by an increase in the abundance of pre-mRNA that lacked a mature 3' end, i.e., that was neither cleaved nor polyadenylated. We infer from these and other data that sequences within the final intron facilitate proper 3'-end formation, possibly through an association with the components of a productive spliceosome.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.