The Cap and the 3′ Splice Site Similarly Affect Polyadenylation Efficiency

Abstract

The majority of pre-mRNAs in mammalian cells are processed in the nucleus by 7-methylguanosine (m 7 GpppG) cap formation on the 5Ј end and splicing and polyadenylation on the 3Ј end. The biochemistry of each of these individual processing reactions has been extensively studied (9,10,13,16,27,29). These studies suggest that in the cell, the processing of an mRNA is highly coordinated and that regulatory interactions must occur between the specialized factors which mediate each individual processing reaction. Such coordination has been suggested in the exon definition model proposed by Berget (2).According to the exon definition model (Fig. 1), mammalian interior exons, because of their small size (average of 137 nucleotides), are defined when the splicing factors locate a pair of closely spaced splice sites in exonic polarity (3Ј splice siteexon sequences-5Ј splice site). This localization of splice sites allows exon definition by the binding of U1 and U2 small nuclear ribonucleoproteins (snRNPs) and associated factors (2). However, the two terminal exons require a different mechanism given that the first exon has a cap structure instead of a 3Ј splice site and the last exon has a polyadenylation signal instead of a 5Ј splice site.The mechanism for the processing of the first exon has been suggested by the work of Izaurralde et al. (9), which showed that an m 7 GpppG cap and a nuclear cap-binding complex (CBC; an 80-kDa and a 20-kDa cap-binding protein) were essential for efficient in vitro removal of the adjacent intron, using a simple one-intron substrate. These data suggest that an early stage of spliceosome assembly, possibly the formation of the commitment complex, is blocked by either the use of a cap analog (to which CBC does not bind) or the depletion of the CBC from splicing extracts. These data suggest that the CBC, in association with the m 7 GpppG cap, may interact with components of the commitment complex (Fig. 1), providing a means to define the exon or to activate the progression to full spliceosome assembly. This effect of the cap and the CBC appears to affect only the removal of the first intron. Previous in vitro experiments using a two-intron system indicated that changing the m 7 GpppG cap to an adenosine inhibited removal of the first intron but had little or no effect on the removal of the second (22).Several lines of experimentation have suggested that processing of the last exon involves interaction with splicing components at the 3Ј splice site and the polyadenylation complex (Fig. 1). Such interactions have been suggested by experiments using a coupled in vitro splicing and polyadenylation system (19,21). These data showed that mutations in the polyadenylation signal which inhibited polyadenylation also caused a decrease in the efficiency of splicing, i.e., removal of the last intron. Likewise, mutations in the 3Ј splice site of the last exon, which inhibit splicing, caused inhibition of polyadenylation. Analogous transfection experiments have provided similar results in vivo (4,17,18). ...

5′-Capping enzymes are targeted to pre-mRNA by binding to the phosphorylated carboxy-terminal domain of RNA polymerase II

“…Our results are consistent with previous studies showing the cap is important for subsequent RNA processing events (Gilmartin et al 1988;Izaurralde et al 1994;Colot et al 1996;Cooke and Alwine 1996;Fresco and Buratowski 1996;Schwer and Shuman 1996). The uncapped RNA fraction in vivo was enriched for unspliced ␤-globin (Fig.…”

“…3, lanes 1,2). These observations are consistent with in vitro studies showing that the cap enhances 3Ј processing (Gilmartin et al 1988;Cooke and Alwine 1996). These experiments strongly imply that RNAs bound by GST-eIF4E have functional caps, whereas unbound RNAs lack cap structures capable of supporting efficient splicing and 3Ј processing.…”

“…The cap appears to earmark an RNA molecule for processes that are specific to Pol II transcripts. It enhances splicing, 3Ј processing, transport, and translation of mRNA (Konarska et al 1984;Edery and Sonenberg 1985;Shatkin 1985;Gilmartin et al 1988;Hamm and Mattaj 1990;Izaurralde et al 1994;Colot et al 1996;Cooke and Alwine 1996). The cap also stabilizes mRNA (Furuichi et al 1977;Green et al 1983); moreover, decapping is important for triggering degradation (Beelman and Parker 1995).…”

“…Previous experiments have not addressed whether capping is also integrated with transcription by a mechanism involving the CTD. Because the 5Ј cap stimulates splicing and polyadenylation (Edery and Sonenberg 1985;Izaurralde et al 1994;Cooke and Alwine 1996), it remains possible that a capping defect could contribute to reduced processing of transcripts made by Pol II with a truncated CTD. In this paper, we show that CTD truncation does, in fact, reduce mRNA capping in vivo, although this effect cannot fully account for the splicing and polyadenylation defects of transcripts made by the truncated Pol II.…”

Identification of two cis-acting elements that independently regulate the length of poly(A) on Xenopus albumin pre-mRNA

“…Regulated polyadenylation in constructs bearing albumin intron 14 and exon 15+ L cells were transfected with a hybrid construct that had the b-globin gene fused to the a9 portion of the albumin gene spanning from the 59 splice site of intron 14 to 212 bp of 39 flanking sequence+ RT-PCR poly(A) analysis was performed on total RNA from transfected cells (lanes 2 and 4) and compared to a control for albumin mRNA in liver (lanes 3 and 5)+ Analyses in lanes 2 and 3 used a radiolabeled primer for intron 14 (open arrowhead, bottom) to measure poly(A) length in pre-mRNA, and those in lanes 4 and 5 used a primer for exon 15 (filled arrowhead, bottom) to measure poly(A) length on both pre-mRNA and fully processed mRNA+ The limit size for products amplified with the intron 14 primer is 193 bp, whereas the limit size for products amplified with the exon 15 primer is 145 bp+ Albumin intron 14 is identified by the asterisk+ Several levels of regulation have been found for premRNA 39 processing+ A number of studies have shown a link between splicing and 39 processing (Niwa et al+, 1990;Niwa & Berget, 1991;Nesic & Maquat, 1994;Cooke & Alwine, 1996), both in vivo and in vitro+ Elements upstream of AAUAAA in a number of viral RNAs can enhance the efficiency of 39 processing+ With these mRNAs, the predominant mode of action appears to be stabilization of CPSF binding (Lutz & Alwine, 1994;Gilmartin et al+, 1995)+ In the case of the 39 processing of the pre-mRNA for U1 snRNP A protein, binding of two molecules of U1A to the 39 UTR of U1A pre-mRNA has Figure 4+ B: Poly(A) length regulation by a sequence in intron 14 or the 59 end of exon 15+ A region spanning the terminal albumin intron (intron 14, asterisk) and the 59 20 bp of exon 15 was cloned into CMV-glo-SPA+ Poly(A) analysis was performed using a primer to globin exon 3 (filled arrowhead) to detect the product bearing spliced intron 14+ C: Identification of a PLE between Ϫ124 and Ϫ104+ The 21 bp spanning Ϫ124/Ϫ104 of albumin exon 15 (shaded) was inserted between the stop codon and the synthetic poly(A) element of CMV-glo-SPA+ The length of poly(A) on the hybrid pre-mRNA expressed in transfected L cells was determined using radiolabeled primers to globin intron 2 (lanes 2 and 3, open arrowhead) or exon 3 (lanes 4 and 5, filled arrowhead)+ no effect on cleavage, but blocks the addition of poly(A) by interacting with poly(A) polymerase (Gunderson et al+, 1994)+ U1A itself interacts with the 160-kDa subunit of CPSF to stabilize the binding of this complex to AAUAAA (Lutz et al+, 1996)+ In fact, adding increasing concentrations of U1A to an in vitro polyadenylation reaction can increase both the number of polyadenylated molecules and the length of added poly(A) (Lutz et al+, 1996)+ However, continued addition of U1A reduces the length of added poly(A)+ Although our data are consistent with regulation of poly(A) addition, we cannot formally rule out the possibility that albumin pre-mRNA receives a .200-nt poly(A) tail and, prior to splicing of the terminal intron, the PLE directs deadenylation to the 17 nt seen here and in our previous study+ Paillard et al+ (1998) identified an element (EDEN) consisting of several U(A/G) repeats in th...…”