Linking Splicing to Pol II Transcription Stabilizes Pre-mRNAs and Influences Splicing Patterns

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

Bridged filaments of histone-like nucleoid structuring protein pause RNA polymerase and aid termination in bacteria.

Bacterial H-NS forms nucleoprotein filaments that spread on DNA and bridge distant DNA sites. H-NS filaments co-localize with sites of Rho-dependent termination in Escherichia coli, but their direct effects on transcriptional pausing and termination are untested. In this study, we report that bridged H-NS filaments strongly increase pausing by E. coli RNA polymerase at a subset of pause sites with high potential for backtracking. Bridged but not linear H-NS filaments promoted Rho-dependent termination by increasing pause dwell times and the kinetic window for Rho action. By observing single H-NS filaments and elongating RNA polymerase molecules using atomic force microscopy, we established that bridged filaments surround paused complexes. Our results favor a model in which H-NS-constrained changes in DNA supercoiling driven by transcription promote pausing at backtracking-susceptible sites. Our findings provide a mechanistic rationale for H-NS stimulation of Rho-dependent termination in horizontally transferred genes and during pervasive antisense and noncoding transcription in bacteria

Death by splicing: tumor suppressor RBM5 freezes splice-site pairing.

In a recent issue of Molecular Cell, Bonnal et al. (2008) demonstrate that the tumor suppressor gene RBM5 regulates alternative splicing of Fas pre-mRNA by interfering with splice-site pairing

Death by splicing: tumor suppressor RBM5 freezes splice-site pairing.

In a recent issue of Molecular Cell, Bonnal et al. (2008) demonstrate that the tumor suppressor gene RBM5 regulates alternative splicing of Fas pre-mRNA by interfering with splice-site pairing

Spliceosome assembly pathways for different types of alternative splicing converge during commitment to splice site pairing in the A complex.

Differential splice site pairing establishes alternative splicing patterns resulting in the generation of multiple mRNA isoforms. This process is carried out by the spliceosome, which is activated by a series of sequential structural rearrangements of its five core snRNPs. To determine when splice sites become functionally paired, we carried out a series of kinetic trap experiments using pre-mRNAs that undergo alternative 5' splice site selection or alternative exon inclusion. We show that commitment to splice site pairing in both cases occurs in the A complex, which is characterized by the ATP-dependent association of the U2 snRNP with the branch point. Interestingly, the timing of splice site pairing is independent of the intron or exon definition modes of splice site recognition. Using the ATP analog ATPgammaS, we showed that ATP hydrolysis is required for splice site pairing independent from U2 snRNP binding to the pre-mRNA. These results identify the A complex as the spliceosomal assembly step dedicated to splice site pairing and suggest that ATP hydrolysis locks splice sites into a splicing pattern after stable U2 snRNP association to the branch point

Spliceosome Assembly Pathways for Different Types of Alternative Splicing Converge during Commitment to Splice Site Pairing in the A Complex▿ †

Differential splice site pairing establishes alternative splicing patterns resulting in the generation of multiple mRNA isoforms. This process is carried out by the spliceosome, which is activated by a series of sequential structural rearrangements of its five core snRNPs. To determine when splice sites become functionally paired, we carried out a series of kinetic trap experiments using pre-mRNAs that undergo alternative 5′ splice site selection or alternative exon inclusion. We show that commitment to splice site pairing in both cases occurs in the A complex, which is characterized by the ATP-dependent association of the U2 snRNP with the branch point. Interestingly, the timing of splice site pairing is independent of the intron or exon definition modes of splice site recognition. Using the ATP analog ATPγS, we showed that ATP hydrolysis is required for splice site pairing independent from U2 snRNP binding to the pre-mRNA. These results identify the A complex as the spliceosomal assembly step dedicated to splice site pairing and suggest that ATP hydrolysis locks splice sites into a splicing pattern after stable U2 snRNP association to the branch point

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

<p>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 (K_m) 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 K_m value indicated below each graph. The dotted line represents the mathematical prediction for the accumulation of spliced mRNA based on the K_m value indicated below each graph. </p

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

<p>(A) Diagrams of the two templates used in the transcription/splicing assay, both contain the first exons of the <i>β-globin</i> 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. </p

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

<p>(A) Diagrams of the wt <i>β-globin</i> 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]). </p

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

<p>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.</p