The poly(A) tract Since post-transcriptional events may represent an important control mechanism in the regulation of genetic expression, we have investigated the possibility of methylation as an additional post-transcriptional modification of mRNA. Earlier work with bacterial and phage mRNA produced strong evidence for the essential absence of methylation in these systems, being no higher than one per 3500 nucleotides (8). Other early studies with mammalian heterogeneous nuclear RNA (HnRNA) indicated that methylation was either nonexistent or very low (9, 10). The discovery of poly-(A) has now made it possible to obtain pure mRNA fractions through affinity chromatography and, therefore, to search for low levels of methylation in mRNA without interference from rRNA contamination. Recently Perry and Kelley reported the existence of methylation in mouse L cell mRNA at about one-sixth the level found in rRNA, and both base and ribose methylations were found (11). This paper reports the existence of methylated nucleosides in the mRNA of Novikoff hepatoma cells and identifies the unique distribution of methylated moieties. A preliminary report of these results has appeared elsewhere (12). METHODSCell Culture and Labeling. The NlS1 strain of Novikoff hepatoma cells was grown in culture in Swimm's S-77 medium supplemented with 4 mM glutamine and 10% (v/v) dialyzed calf serum (13

The general splicing factor SF2/ASF binds in a sequence-specific manner to a pm~ne-dch exonic splicing enhancer {ESE) in the last exon of bovine growth hormone (bGH) pre-mRNA. More importantly, SF2/ASF stimulates in vitro splicing of bGH intron D through specific interaction with the ESE sequences. However, another general splicing factor, SC35, does not bind the ESE sequences and has no effect on bGH intron D splicing. Thus, one possible function of SF2/ASF in alternative and, perhaps, constitutive pre-mRNA splicing is to recognize ESE sequences. The stimulation of bGH intron D splicing by SF2/ASF is counteracted by the addition of hnRNP A1. The relative levels of SF2/ASF and hnRNP A1 influence the efficiency of bGH intron D splicing in vitro and may be the underlying mechanism of this alternative pre-mRNA processing event in vivo.[Key Words: Alternative splicing; bovine growth hormone pre-mRNA; SF2/ASF; hnRNP A1; exonic splicing enhancer]Received September 3, 1993; accepted October 6, 1993.Pre-mRNA splicing requires accurate selection of 5' and 3' splice sites. For many genes, alternative splice sites can be selected from a single primary transcript to generate two or more mRNAs encoding multiple protein isoforms. Although alternative pre-mRNA splicing provides an important route for the regulation of gene expression, the underlying regulatory mechanisms remain largely uncharacterized.A central question in constitutive and alternative premRNA splicing concems the selection of specific splice sites. Introns are defined by the presence of a conserved 5' splice site, a 3' splice site, a polypyrimidine tract, and a less well-conserved branch site. Although these signals are required for splicing, they are insufficient to specify all introns. Several studies have shown that exon sequences can also play an important role in splice site Hoshijima et al. 1991}. The trans-acting factors encoded by the transformer (tra) and transformer 2 (tra2) genes together activate the 3' splice site upstream of the dsx female-specific exon 4 through interaction with six 13-nucleotide repeats present in the exon (Tian and Maniatis 1992). Thus, protein factors are capable of binding to exon sequences in a sequence-specific manner and affecting splicing of introns located some distance away from the protein-binding sites.A number of factors required for constitutive splicing have been shown to be involved in alternative splice site selection. The product of the Sex-lethal (Sxl) gene regulates alternative splicing of tra pre-mRNA by repressing the inclusion of exon 2 (Boggs et al. 1987). Recent results showed that Sx/inhibits the use of the 3' splice site upstream of exon 2 by blocking the binding of the essential splicing factor U2AF to the polypyrimidine tract (Valcarcel et al. 1993). Binding of U1 small nuclear ribonucleoprotein (snRNP) to a pseudo-5' splice site, as opposed to the authentic 5' splice site, has been implicated in the regulated alternative intron retention of Drosophila P-element pre-mRNA in somatic cells (Siebel et ...

An analysis of the methylated constituents of L cell mRNA by a combination of chromatographic methods and enzymatic treatments indicates that they comprise both 2'-O-methyl nucleosides and N6-methyl adenine, and/or 1-methyl adenine, and suggests that the 2'-O-methyl nucleotides, Ym, are part of an unusual class of sequences forming the 5' terminus of mRNA. These sequences seem to contain two 2'-O-methyl residues and a terminal residue that is not phosphorylated but, nevertheless, is blocked with respect to polynucleotid kinase reactivity. A strong candidate is a sequence of the type XppY1mpY2mpZp..., where X represents a blocking group which is itself occasionally methylated. The sequences isolated from total poly(A)+ mRNA contain all four species of 2'-O-methylated nucleoside, indicating some variability among different mRNA species. The methylated sequences do not appear to be enriched in the mRNA which hybridizes with repetitive DNA. The average L cell mRNA molecule also contains three residues of N6-methyl adenine. These residues are not part of the poly(A) segment, but appear to be located internal to the poly(A) near the 3' end of the mRNA molecules.

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