The P-Loop Domain of Yeast Clp1 Mediates Interactions Between CF IA and CPF Factors in Pre-mRNA 3′ End Formation

Cleavage factor IA (CF IA), cleavage and polyadenylation factor (CPF), constitute major protein complexes required for pre-mRNA 3′ end formation in yeast. The Clp1 protein associates with Pcf11, Rna15 and Rna14 in CF IA but its functional role remained unclear. Clp1 carries an evolutionarily conserved P-loop motif that was previously shown to bind ATP. Interestingly, human and archaean Clp1 homologues, but not the yeast protein, carry 5′ RNA kinase activity. We show that depletion of Clp1 in yeast promoted defective 3′ end formation and RNA polymerase II termination; however, cells expressing Clp1 with mutant P-loops displayed only minor defects in gene expression. Similarly, purified and reconstituted mutant CF IA factors that interfered with ATP binding complemented CF IA depleted extracts in coupled in vitro transcription/3′ end processing reactions. We found that Clp1 was required to assemble recombinant CF IA and that certain P-loop mutants failed to interact with the CF IA subunit Pcf11. In contrast, mutations in Clp1 enhanced binding to the 3′ endonuclease Ysh1 that is a component of CPF. Our results support a structural role for the Clp1 P-loop motif. ATP binding by Clp1 likely contributes to CF IA formation and cross-factor interactions during the dynamic process of 3′ end formation

ATP binding status of recombinant CF IA factors.

<p>ATP binding status of recombinant CF IA factors.</p

Gene expression in strains expressing Clp1 proteins carrying a mutant P-loop.

<p>(A) Northern analysis of total RNAs obtained from wild-type and <i>CLP1</i> D161A and <i>CLP1</i> K136A T137A mutant strains following growth in YPD. Probes were as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029139#pone-0029139-g001" target="_blank">figure 1C</a>. (B) qRT-PCR analysis of candidate mRNAs that were identified during gene expression profiling of <i>CLP1</i> D161A and <i>CLP1</i> K136A T137A mutant strains. Transcript levels are presented relative to wild-type <i>CLP1</i>, which was fixed at 100%. Data shown are the mean of three independent biological replicates and error bars indicate standard deviation.</p

Effects of Clp1 depletion on gene expression.

<p>(A) Schematic presentation of the Clp1 fusion gene, which is expressed under the control of <i>GAL10</i> promoter. Encoded are an amino-terminal ubiquitin moiety fused in frame to Clp1 together with an HA-tag. Removal of the ubiquitin moiety by cellular deubiquitylases results in a protein with an arginine (R) at the amino-terminus creating an unstable protein that is targeted for degradation by the proteasome. A strain carrying this fusion gene is viable on YPGal medium but not on YPD, which contains repressive glucose as sole carbon source. (B) Western Blot of total extracts obtained from wild-type and <i>GAL-UBI-R-HA-CLP1</i> expressing strains following growth in YPD for the indicated times. Western blots were decorated with antibody against the HA-tag. Ponceau S staining served as control for equal loading. (C) Northern analysis of total RNAs obtained from wild-type and <i>GAL-UBI-R-HA-CLP1</i> expressing strains following growth in YPD for the indicated times. RNAs were detected with random prime labeled probes directed against the open reading frames as indicated on the left of each panel. The asterisk in the first panel marks a likely degradation product of the <i>UBI-R-HA-CLP1</i> mRNA. In <i>CYH2</i> and <i>NRD1</i> panels ‘ext’ denotes 3′ extended transcripts. 18S rRNA was detected with an end-labeled oligonucleotide and was used as control for equal loading. Schematically depicted on the right are features of analyzed and neighbouring genes with arrows indicating sites of 3′ end cleavage and polyadenylation.</p

Coupled <i>in vitro</i> transcription/3′ end processing.

<p>(A) Western blot analysis of CF IA factors partially purified from strains expressing ProtA-Clp1 and ProtA-Clp1 K136A T137A, respectively. Factor purification included a high salt wash (1 M KCl) of bound material to probe stability and integrity of the protein complexes. Decreasing amounts of CF IA associated with ProtA-Clp1 or ProtA-Clp1 K136A T137A were analyzed for the presence of the four CF IA subunits Rna15, Pcf11, Clp1 and Rna14 as indicated on the right. (B) Schematic presentation of the transcription template that was used in transcription/3′ end processing reactions <i>in vitro</i>. The construct includes Gal4 binding sites, a <i>CYC1</i> promoter and five G-less cassettes; the length of the cassettes in nucleotides is indicated. The <i>CYC1</i> terminator has been inserted between the 100 and 120 cassettes; also indicated are specific sequence elements (EE: efficiency element; PE: positioning element; UUE: upstream U-rich element; DUE: downstream U-rich element; and the poly(A) site). <i>In vitro</i> transcription produces a 0.5 kb polyadenylated RNA and read-through transcription produces a RNA of more than 1.3 kb length. (C) Western analysis of ProtA-Rna15 expressing whole cell extracts before and after two consecutive rounds of depletion on IgG-agarose. Increasing amounts (1, 2 and 4 µl) of extract (XT) and of depleted extract (2× depl) were resolved by SDS-PAGE and following transfer on PVDF membrane ProtA-Rna15 was detected using an anti-HA-HRP secondary antibody that readily bound to the ProteinA moiety of the fusion protein. Ponceau S staining of a section of the membrane is shown to demonstrate comparable loading between extract and depleted extract. (D) <i>In vitro</i> transcription/3′ end processing reactions with CF IA depleted extracts in with or without adding back ProtA-Clp1 and ProtA-Clp1 K136A T137A purified CF IA factors as indicated. On the top of each panel is indicated the salt concentration (mM KCl) applied in the final wash step of the protocol that was applied to purify CF IA factors associated with ProtA-tagged wild-type and mutant Clp1. G-less transcripts were separated on 8.3 M Urea 6% polyacrylamide gels. Migration of the G-less transcripts is indicated on the left. Gels at the bottom of each panel show the analysis of pre-mRNA 3′ end cleavage. Non-radioactive <i>in vitro</i> transcription reactions were performed and obtained RNAs were subjected to a ligation-mediated RT-PCR procedure. Formation of PCR product correlates with 3′ end cleavage activity and encompasses the site of poly(A) addition. (E) Quantification of the gels shown in (D). The obtained signals were normalized to uridine-content of the cassettes and presented as percentage of G-less transcription relative to the 100 cassette that is placed immediately upstream of the <i>CYC1</i> terminator; the site of poly(A) addition has been fixed at “0” bp. Termination and 3′ end formation is reflected by strength of transcription downstream of the terminator, and is indicated by 120/100, 131/100 and 145/100 signal ratios.</p