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RNA polymerase II pauses and associates with pre-mRNA processing factors at both ends of genes - PubMed

RNA polymerase II pauses and associates with pre-mRNA processing factors at both ends of genes

Kira Glover-Cutter et al. Nat Struct Mol Biol. 2008 Jan.

Abstract

We investigated co-transcriptional recruitment of pre-mRNA processing factors to human genes. Capping factors associate with paused RNA polymerase II (pol II) at the 5' ends of quiescent genes. They also track throughout actively transcribed genes and accumulate with paused polymerase in the 3' flanking region. The 3' processing factors cleavage stimulation factor and cleavage polyadenylation specificity factor are maximally recruited 0.5-1.5 kilobases downstream of poly(A) sites where they coincide with capping factors, Spt5, and Ser2-hyperphosphorylated, paused pol II. 3' end processing factors also localize at transcription start sites, and this early recruitment is enhanced after polymerase arrest with the elongation factor DRB. These results suggest that promoters may help specify recruitment of 3' end processing factors. We propose a dual-pausing model wherein elongation arrests near the transcription start site and in the 3' flank to allow co-transcriptional processing by factors recruited to the pol II ternary complex.

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Figures

Figure 1
Figure 1. Capping Enzymes localize with paused pol II at 5′ and 3′ ends and throughout the p21 gene

Diagram of the p21 gene with the center of real-time PCR products marked relative to the transcription start site as in ref. . Relative ChIP signals in HCT116 cells plotted for A) total pol II and CTD Ser2-PO4, B) cap methyltransferase (MT) C) human capping enzyme (HCE) and D) Spt5 before (untreated, dark bars) and after activation by Nutlin-3a (pale bars). Down arrow and * indicates poly(A) signal at +8570. Flanking primers −2965 and +11443 serve as intergenic background controls. Pol II traces from A are included in B-D. Values are normalized to the maximal value in the Nutlin-3a data set. For reasons we do not understand, the density of capping enzymes relative to pol II and Spt5 was reproducibly higher at amplicon +1775 in intron 1 than at other positions. Mean values from n PCR reactions (n) with standard errors of mean (SEM) are shown.

Figure 2
Figure 2

A) Pol II pausing and histone localization at 5′ and 3′ ends of p21. Pol II, Spt5, histone H3 and TFIIB localization by ChIP on the Nutlin-3a activated p21 gene. Displacement of H3 occurs at sites of pol II accumulation at the 5′ and 3′ ends. Note that unlike pol II and Spt5, TFIIB peaks near the 5′ end but not at the 3′ end. B) DRB inhibits pausing and accumulation of MT downstream of the p21 poly (A) site. ChIP of pol II and MT after induction by Nutlin-3a or DRB normalized to the maximum value in each data set to emphasize differences in 5′-3′ distribution. Mean values from n PCR reactions with standard errors of mean (SEM) are shown.

Figure 3
Figure 3. Capping Enzymes track with pol II on c-Myc and GAPDH genes

A) Positions of real-time PCR products are indicated relative to the c-Myc P2 start site. Relative ChIP signals are shown for total pol II, CTD Ser2-PO4, and Spt5 B) MT and HCE on c-Myc in HCT116 cells. Traces of pol II and Spt5 from A serve as reference. C) total pol II, CTD Ser2-PO4, and Spt5 on GAPDH. D) MT and HCE on GAPDH. Traces of pol II and Spt5 from C serve as reference. * and down arrow indicate poly(A) signals at +5166 relative to the c-Myc P2 transcription start site, and +3830 for GAPDH. Mean values from n PCR reactions with standard errors of mean (SEM) are shown..

Figure 4
Figure 4

A, B. Association of capping factors with DRB-arrested pol II complexes on GAPDH. Positions of real-time PCR products are indicated. Relative ChIP signals are shown for pol II, MT and HCE on the GAPDH gene in untreated and DRB treated HCT116 cells. C, D. Localization of capping factors, pol II and Spt5 on histone H4 and U2 snRNA genes. C) pol II, Spt5 MT and HCE on histone H4/d (Accession no. X60483). D) pol II, Spt5, MT, HCE and CBC on U2 snRNA (Accession no. U57614). CBC data is the sum of 4 determinations each with anti-CBC20 and 80. Mature RNA 3′ ends (down arrow and *) map to +387 for H4/d and +190 for U2. Mean values from n PCR reactions with standard errors of mean (SEM) are shown.

Figure 5
Figure 5. Localization of 3′ end processing factors at 5′ and 3′ ends of p21, c-Myc and GAPDH

Positions of real-time PCR products on each gene are indicated. Relative ChIP signals in HCT116 cells are shown for A) CPSF73 and CstF77 on c-Myc and B) GAPDH. C) CstF77 and D) CPSF73 on untreated and Nutlin-3a activated p21 normalized to the maximum value for the Nutlin-3a data set. * and down arrow indicate poly(A) sites. Mean values from n PCR reactions with standard errors of mean (SEM) are shown.

Figure 6
Figure 6

A) Co-transcriptional recruitment of 3′ end processing factors to a histone gene. ChIP of CPSF73 and CstF77 subunits of HLF and pol II on histone H4/d in HCT116 cells. Down arrow and * indicates mRNA 3′ end. B, C) DRB enhances recruitment of 3′ end processing factors at the 5′ end of GAPDH. Relative ChIP signals are shown for B) CPSF73 and C) CstF77 on GAPDH untreated (un) and after DRB treatment. Pol II and CstF77 profiles for untreated cells are the same as Fig. 5B. The untreated CPSF73 data is a subset of that in Fig. 5B. Data in B are normalized to the maximum value for the DRB sample at +55, and in C they are normalized to the maximum value for the untreated sample at +4511. Mean values from n PCR reactions with standard errors of mean (SEM) are shown.

Figure 7
Figure 7. Dual pausing model for co-transcriptional 5′ and 3′ end maturation of polyadenylated mRNAs

This model is based on data reported here and in references ,.

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