Cap-dependent RNA splicing in a HeLa nuclear extract.

Abstract

We have studied the involvement of the 5' cap structure in the splicing of precursor mRNAs in a HeLa nuclear extract. We show that precursor mRNAs are spliced efficiently only when they possess a cap structure and that preincubation of a HeLa nuclear extract rendered the splicing reaction highly sensitive to inhibition by cap analogues. This sensitization was dependent on exogenous Mg2' but not exogenous ATP or GTP.These results demonstrate that splicing in a nuclear extract is highly dependent on the cap structure as was demonstrated for the splicing process in a HeLa whole-cell extract [Konarska, M. M., Padget, R. A. & Sharp, P. A. (1984) Cell 38,[731][732][733][734][735][736], and thus support the contention that cap recognition is an important feature of eukaryotic mRNA biogenesis.All eukaryotic cellular mRNAs analyzed to date are blocked at their 5' terminus by the cap structure, m7G(5')ppp(5')N (1). There is considerable evidence that the cap structure enhances translational efficiency by facilitating ribosome binding to mRNA (1). This interaction is mediated by a distinct group of proteins defined as cap-binding proteins (2,3).With the advent of efficient in vitro splicing systems (4-10), two recent studies have suggested that the cap structure may play a significant role in the splicing process as well (6, 7). Using a HeLa nuclear extract, Krainer et al. (7) were able to show efficient splicing of in vitro synthesized, truncated human P-globin transcripts. They showed that although uncapped transcripts could still be spliced, the efficiency was one-half to one-third that for their enzymatically capped counterparts. In addition, splicing of uncapped precursor mRNAs (pre-mRNAs) The transcripts were stored at -70'C in water and used directly in the splicing reaction.In Vitro Splicing Reaction. HeLa nuclear extracts were prepared as described (13). All experiments described throughout were performed using the same extract prepara- RESULTSTo characterize the splicing activity of our HeLa nuclear extracts, we used the plasmid pSP64-il/3A6. This plasmid and its use in the nuclear splicing system were described by Krainer et al. (7) and Ruskin et al. (8). For preparation of 7590The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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

“…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.…”

The coat protein of the yeast double-stranded RNA virus L-A attaches covalently to the cap structure of eukaryotic mRNA.

“…It is of particular importance in translation, at the initiation level (see references 14 and 51 for reviews). The cap also facilitates mRNA splicing (15,28) and 3' end processing (19,25) and is required for nucleocytoplasmic RNA transport (24). In addition, the cap protects mRNA against 5'-3' exonucleolytic degradation (18,21).…”

Effect of the cap structure on pre-mRNA splicing in Xenopus oocyte nuclei.

“…Since in vitro splicing systems were developed, considerable progress has been made in understanding the mechanism of nuclear precursor mRNA (premRNA) splicing (for review, see Green 1986;Padgett et al 1986;Sharp 1987). A cap structure, m^G(5')ppp(5')N, blocking the 5' termini of many eukaryotic mRNAs has been shown to play an important role in pre-mRNA splicing (Krainer et al 1984;Konarska et al 1984;Edery and Sonenberg 1985;Ohno et al 1987). Although the requirement for the cap appears to differ slightly between whole-cell and nuclear extracts (Konarska et al 1984: Edery andSonenberg 1985), the results obtained with the in vitro splicing reaction indicate that cap recognition is an important event in the early process of the splicing reaction.…”

“…A cap structure, m^G(5')ppp(5')N, blocking the 5' termini of many eukaryotic mRNAs has been shown to play an important role in pre-mRNA splicing (Krainer et al 1984;Konarska et al 1984;Edery and Sonenberg 1985;Ohno et al 1987). Although the requirement for the cap appears to differ slightly between whole-cell and nuclear extracts (Konarska et al 1984: Edery andSonenberg 1985), the results obtained with the in vitro splicing reaction indicate that cap recognition is an important event in the early process of the splicing reaction. By use of pre-mRNAs that contain three exons and two introns, we have shown that when a pre-mRNA is capped, the upstream intron is spliced out more efficiently than the downstream intron because of the differential effect of the cap structure on the two introns.…”