Linking splicing to Pol II transcription stabilizes pre-mRNAs and influences splicing patterns - PubMed (original) (raw)

Linking splicing to Pol II transcription stabilizes pre-mRNAs and influences splicing patterns

Martin J Hicks et al. PLoS Biol. 2006 Jun.

Abstract

RNA processing is carried out in close proximity to the site of transcription, suggesting a regulatory link between transcription and pre-mRNA splicing. Using an in vitro transcription/splicing assay, we demonstrate that an association of RNA polymerase II (Pol II) transcription and pre-mRNA splicing is required for efficient gene expression. Pol II-synthesized RNAs containing functional splice sites are protected from nuclear degradation, presumably because the local concentration of the splicing machinery is sufficiently high to ensure its association over interactions with nucleases. Furthermore, the process of transcription influences alternative splicing of newly synthesized pre-mRNAs. Because other RNA polymerases do not provide similar protection from nucleases, and their RNA products display altered splicing patterns, the link between transcription and RNA processing is RNA Pol II-specific. We propose that the connection between transcription by Pol II and pre-mRNA splicing guarantees an extended half-life and proper processing of nascent pre-mRNAs.

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Figures

Figure 1

Figure 1. Linking Splicing to Pol II Transcription Increases the Efficiency of Pre-mRNA Splicing

(A) Diagrams of the two templates used in the transcription/splicing assay, both contain the first exons of the_β-globin_ gene. The template on the left is headed by the AdML Pol II promoter while the right template is headed by the T7 promoter. (B) Representative autoradiograms of the in vitro transcription/splicing assay appear below the diagrams. Pre-mRNA transcripts were either synthesized by Pol II (left) or T7 polymerase (right). Pre-mRNA transcript, spliced product, and intermediate lariat bands are indicated. (C) Quantitation of the data in (B). Molar concentration of RNA was determined by normalizing counts per min to a known concentration of radiolabeled RNA.

Figure 2

Figure 2. The Link between Pol II Transcription and Splicing Increases the Stability of Nascent Transcripts but Not the Rate of Splicing

(A) Diagrams of the wt_β-globin_ minigene DNA template with the Pol II promoter (top), the T7 promoter (middle), or the pre-synthesized RNA transcript (lower). (B) Representative autoradiograms show the time course of the transcription/splicing UTP chase experiment for Pol II- (left) or T7- (middle) generated transcripts, and the splicing profile of pre-synthesized pre-mRNAs (right). For the transcription/splicing UTP chase experiment, the zero time point refers to the initiation of the chase reaction through the addition of excess unlabeled UTP. Pre-mRNA transcript, spliced product, and intermediate lariat bands are indicated. The pre-synthesized pre-mRNA (right) is capped. (C) Quantitation of the data in (B) by computing the fraction spliced ([lariat] + [product])/ ([lariat] + [product] + [pre-mRNA]). (D) Quantitation of transcript degradation ([lariat] + [product] + [pre-mRNA]).

Figure 3

Figure 3. Functional Splice Sites Increase the Stability of Pol II Transcripts

(A) Diagrams of DNA templates for wt (top) and mutant_β-globin_ minigenes. The X indicates abolished splice sites. (B) Representative autoradiograms of the transcription/splicing chase experiments for the wt substrate (left), the double 5′ and 3′ splice site mutant (ss M) (middle), the mutant substrate (middle), and the wt substrate in extracts depleted of functional U2 snRNA (right). (C) Average rates of degradation observed for the Pol II transcripts wt, 5′ ss M, 3′ ss M, the double 5′ and 3′ ss M, wt substrate in U2 snRNA depleted extract, and wt substrate in U5 snRNA depleted extract.

Figure 4

Figure 4. Minimal Kinetic Scheme of the In Vitro Reaction Linking Pol II Transcription with Pre-mRNA Splicing

The diagram shows a series of dissociation and association constants for the formation of complexes between the DNA template and the transcription machinery, between the pre-mRNA and ribonucleases or the splicing machinery, and between the spliced mRNA and ribonucleases. The catalytic rate constants describe the conversion of NTPs to pre-mRNA for transcription, the conversion of pre-mRNA to mRNA for splicing, and the conversion of pre-mRNA or mRNA to nucleotide monophosphate for ribonuclease digestion.

Figure 5

Figure 5. The Link between Pol II Transcription and Pre-mRNA Splicing Increases the Affinity of Splicing Factors to the Nascent Pre-mRNA

A mathematical model is used to describe the basic reactions involved in the coupled transcription/splicing reaction. The splicing profiles obtained from RNAs generated by Pol II (A) or by T7 polymerase (B) were fit to the mathematical prediction by testing various affinities (Km) between the splicing machinery and the pre-mRNA. (A and B) represent the best fit for either reaction. Squares represent pre-mRNA and triangles represent spliced mRNA. The solid line represents the mathematical prediction for the accumulation of pre-mRNA based on the Km value indicated below each graph. The dotted line represents the mathematical prediction for the accumulation of spliced mRNA based on the Km value indicated below each graph.

Figure 6

Figure 6. The Link between Pol II Transcription and Splicing Influences Alternative 3′ Splice-Site Choice

(A) Diagrams of the_wt14_ minigenes with duplicated 3′ splice sites, either as a DNA template preceded by a Pol II promoter (left) or the pre-synthesized RNA transcript (right). The proximal exon (exon 2A) does not contain a 5′ splice site, is significantly shorter than the distal exon, and lacks exonic splicing enhancers. (B) Representative autoradiograms show the time course of the transcription/splicing UTP chase experiment (left) and the splicing profile of pre-synthesized RNA transcripts (right). The pre-mRNA transcript, spliced products, and intermediate lariat bands are indicated. (C) Quantitation of distal and proximal spliced product formation from (B).

Comment in

References

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