Inhibition of splicing but not cleavage at the 5' splice site by truncating human beta-globin pre-mRNA.

Abstract

Human P-globin mRNAs truncated in the second exon or in the first intron have been processed in vitro in a HeLa cell nuclear extract. Transcripts containing a fragment of the second exon as short as 53 nucleotides are efficiently spliced, whereas transcripts truncated 24 or 14 nucleotides downstream from the 3' splice site are spliced inefficiently, if at all. AU of these transcripts, however, are efficiently and accurately cleaved at the 5' splice site. In contrast, RNA truncated in the first intron, 54 nucleotides upstream from the 3' splice site, is not processed at all. These findings suggest that cleavage at the 5' splice site and subsequent splicing steps-i.e., cleavage at the 3' splice site and exon ligation-need not be coupled. Anti-Sm serum inhibits the complete splicing reaction and cleavage at the 5' splice site, suggesting involvement of certain ribonucleoprotein particles in the cleavage reaction. ATP and Mg2' are required for cleavage at the 5' splice site at concentrations similar to those for the complete splicing reaction.Recently, several systems for in vitro splicing of eukaryotic mRNA have been developed. The early systems relied either on coupled transcription and splicing of mRNA transcripts in whole cell extracts from HeLa cells (1-3) or on the addition of exogenous precursor mRNA to whole cell extracts from HeLa cells (4) or to nuclear extracts from MOPC-315 cells (5, 6). Progress was slow due to the inefficiency of the splicing extracts and the limited availability of substrate RNA. The recent development of a highly efficient nuclear splicing extract (7,8) and the methods to transcribe large amounts of precursor RNA from DNA templates cloned under the SP6 promoter (9, 10) led to rapid advances in this field.The steps in mRNA splicing, formulated by recent in vitro studies (refs. 11-16; see ref. 17 for a recent review), appear to be as follows: a capped RNA transcript is cleaved at the 5' splice site and, simultaneously, the first nucleotide of the intron attacks an adenosine residue located 20-40 nucleotides (nt) upstream from the 3' splice site, forming a lariat structure. This lariat intermediate is then cleaved at the 3' splice site with concomitant ligation of the 5' exon, thus generating a spliced mRNA and the free intron in the lariat form. The details of the phosphodiester bond formation during splicing have been determined (13) and the participation of U1 ribonucleoprotein (U1-RNP) in the splicing reaction has been convincingly shown (18)(19)(20)(21).In spite of this remarkable progress, many aspects of the splicing reaction are still poorly understood. The mechanisms of splice site selection and alternative splicing are unknown as are the properties of the partially purified splicing enzymes (22). The events that occur during a pronounced initial lag period that is observed in vitro (7) MATERIALS AND METHODS DNA templates were prepared by linearizing the plasmid pSP64HbA6 (7) with restriction enzymes that cleave within the second exon and the first intron of the human...

The length of the downstream exon and the substitution of specific sequences affect pre-mRNA splicing in vitro.

“…We have shown previously that efficient removal of intron 1 of the human ,B-globin premRNA can be dramatically reduced in vitro by truncating the transcript so that only 14 nucleotides of the second exon remain. This transcript is able to undergo the first step in splicing, i.e., cleavage at the 5' splice site and lariat formation, but is unable to efficiently generate the final spliced product or the released lariat intron (7). Similar observations have also been reported by Ruskin and Green (24).…”

Extensive structural differences between genes for the α1 and α2 chains of type IV collagen despite conservation of coding sequences

“…This hypothesis cannot be rejected in the case of the mismatched introns in the o~(IV) and ce2(IV) genes. Actually, it has been shown that at least in the fl-globin gene exons smaller than 24 if at all [44]. This finding cannot be generalized, however, as an 11 bp long exon containing a part of the sequence coding for the signal peptide cleavage site has been identified in the chicken procr2(I) collagen gene [45].…”

Identification and characterization by antisense oligonucleotides of exon and intron sequences required for splicing.

“…Although it has been known that both the sequences (6,12,14,35,41,49,51) and the lengths (2,9,13,14,34,44,48,51) of exons are important determinants in the mechanism of splice site selection, the details of their involvement are not well understood. We have presented here a method for probing exon sequences with 2'-O-methyl oligoribonucleotides in search of the signals required for splice site selection.…”