Serine-arginine (SR)-rich splicing factors have an exon-independent function in pre-mRNA splicing - PubMed (original) (raw)

Serine-arginine (SR)-rich splicing factors have an exon-independent function in pre-mRNA splicing

K J Hertel et al. Proc Natl Acad Sci U S A. 1999.

Abstract

Two distinct functions have been proposed for the serine-arginine (SR)-rich family of splicing factors. First, SR proteins are essential splicing factors and are thought to function by mediating protein-protein interactions within the intron during spliceosome assembly. Second, SR proteins bind to exonic enhancer sequences and recruit spliceosome components to adjacent introns. The latter activity is required for splice-site recognition and alternative splicing. Until now it has not been possible to determine whether the requirement for SR proteins in the basic splicing reaction is a secondary consequence of their exon-dependent recruitment function. Here we show that RNA substrates containing only 1 nt of exon sequence can undergo the first step of the splicing reaction in vitro and that this activity requires SR proteins. Thus, we provide direct evidence that SR proteins have both exon-independent and exon-dependent functions in pre-mRNA splicing.

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Figures

Figure 1

Figure 1

As few as 3 exon nucleotides are required for the first step of the splicing reaction. (A) Substrates derived from the AdML pre-mRNA lack a downstream exon and the 3′ splice site. (B) Efficient intron lariat formation was observed for substrates containing three or more exon-1 nucleotides during a 1-hr incubation in nuclear extracts.

Figure 2

Figure 2

A single exon nucleotide is required for the first step of the splicing reaction. (A) Comparison of intron lariat formation efficiency for substrates containing 3, 2, or 1 exon nucleotide. (B) Histogram quantifying the data in A.

Figure 3

Figure 3

SR proteins are required for the first step of the splicing reaction with RNA substrates containing 3, 2, or 1 exon nucleotide. (A) The SR protein 9G8 (200 nM) can complement S100 extract-dependent lariat formation of AdML RNA substrates containing 3, 2, or 1 exon nucleotide. (B) The SR proteins SC35 (500 nM), ASF/SF2 (200 nM), and a preparation of total SR proteins from nuclear extracts (100 ng/25 μl reaction) can also complement S100-dependent lariat formation of an AdML minimal substrate containing 3 exon-1 nucleotides. (C and D) The SR protein 9G8 (200 nM) complements S100 extracts to reconstitute both steps of splicing for exon-containing substrates and the first step of splicing for minimal substrates derived from two other pre-mRNAs, ftz (C) and β-globin (D).

Figure 4

Figure 4

A functional RNA-binding domain is required for maximal efficiency of SR-mediated lariat formation with a minimal exon substrate. S100 extracts were complemented with 9G8 (200 nM), a 9G8 hybrid protein (9G8RS) containing the RNA-binding domain of the bacteriophage MS2 in place of the natural RRM (2,000 nM) (42), SF2/ASF (200 nM), SF2/ASF (FF-DD) containing a double Phe-to-Asp mutation within the RRM (800 nM) (43), and an SF2/ASF hybrid protein (SF2/ASFRS) containing a portion of gyrase in place of the RRM (2,000 nM). (B) Histogram showing the quantitation of the data in A.

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