Ssu72 protein mediates both poly(A)-coupled and poly(A)-independent termination of RNA polymerase II transcription

Abstract

Termination of transcription by RNA polymerase II (Pol II) is a poorly understood yet essential step in eukaryotic gene expression. Termination of pre-mRNA synthesis is coupled to recognition of RNA signals that direct cleavage and polyadenylation of the nascent transcript. Termination of nonpolyadenylated transcripts made by Pol II in the yeast Saccharomyces cerevisiae, including the small nuclear and small nucleolar RNAs, requires distinct RNA elements recognized by the Nrd1 protein and other factors. We have used genetic selection to characterize the terminator of the SNR13 snoRNA gene, revealing a bipartite structure consisting of an upstream element closely matching a Nrd1-binding sequence and a downstream element similar to a cleavage/polyadenylation signal. Genome-wide selection for factors influencing recogniton of the SNR13 terminator yielded mutations in the gene coding for the essential Pol II-binding protein Ssu72. Ssu72 has recently been found to associate with the pre-mRNA cleavage/polyadenylation machinery, and we find that an ssu72 mutation that disrupts Nrd1-dependent termination also results in deficient poly(A)-dependent termination. These findings extend the parallels between the two termination pathways and suggest that they share a common mechanism to signal Pol II termination.

FIG. 1.

The SNR13 3′-flanking region contains tandem Nrd1-dependent 3′-end formation elements. (A) Schematic illustration of the ACT-CUP reporter gene containing the SNR13 3′-end formation element, and effects of the insert on transcription and copper sensitivity. Transcription of the fusion gene is driven by the TDH3 gene promoter (TDH3p), and the PGK1 gene polyadenylation signal (PGK1pA) is present downstreamof the CUP1 coding region. (B) cis-acting mutations in the SNR13 NRE that confer copper-resistant growth. Each line represents a unique allele that has one to four point mutations. All the alleles except no. 3 have mutations in both regions I and II, indicated by heavy underlining. The smaller fragments tested in the copper sensitivity assay in panel E are indicated below the sequence of the 125-232 fragment. (C) Copper sensitivity assay of three alleles bearing the U152C mutation. Tenfold serial dilutions (10⁵ to 10¹ cells) of strains containing the indicated alleles were spotted on −Leu plates containing CuSO₄ at the concentrations shown. (D) Comparison of the region I sequence to a portion of the U6R* NRE. Nucleotides at which point mutations conferring copper resistance have been isolated are boldfaced and underlined. (E) Copper sensitivity assay with SNR13 subfragments containing only region I (125-182) or region II (150-232).

FIG. 2.

ssu72 mutants defective for SNR13 3′-end formation. (A) Sequences of an amino-terminal portion of the Ssu72 protein and homologues identified by BLAST searches, showing the identity of the ssu72-G33A and ssu72-G42V alleles. Residues identical to yeast Ssu72 are boxed. S.c., S. cerevisiae; S.p., S. pombe; N.c., Neurospora crassa; A.t., Arabidopsis thaliana; D.m., Drosophila melanogaster; M.m., Mus musculus; H.s., Homo sapiens; C.e., Caenorhabditis elegans; A.g., Anopheles gambiae; E.c., Encephalitozoon cuniculi. (B) Copper sensitivity assay of ssu72-G33A and nrd1-5 mutants harboring ACT-CUP reporter genes with the indicated SNR13 3′-end formation elements. (C) Schematic illustration of the SNR13 chromosomal locus, including the adjacent gene, TRS31. Lines labeled RNA indicate transcripts that accumulate in wild-type and ssu72 strains, and the position of the oligonucleotide probe used in panel D is shown. (D) Northern blot analysis of transcripts from the endogenous SNR13 locus in wild-type and ssu72-G33A strains, before and after a shift to the restrictive temperature (37°C) for 1, 4, or 7 h.

FIG. 3.

General requirement for SSU72 in the Nrd1-dependent 3′-end formation pathway. (A) Copper sensitivity assay of nrd1-5 and ssu72-G33A mutants harboring an ACT-CUP reporter gene with an SNR14 3′-end formation element in the intron. (B) Northern blot analysis of Nrd1 mRNA in wild-type and ssu72-G33A strains at permissive (25°C) and restrictive (37°C for 1 or 4 h) temperatures. Scr1 is the signal recognition particle RNA and is synthesized by Pol III.

FIG. 4.

ssu72-G33A does not affect recognition of the CYC1 cleavage/polyadenylation site. (A) Schematic illustration of the CYC1 gene and 3′-flanking region showing the 83-bp CYCpAmin fragment inserted into the ACT-CUP reporter gene intron. Also shown are the CYCpAmax and CYCds fragments used in the TRO experiments for which results are shown in Fig. 5. (B) Copper sensitivity assay with wild type, nrd1-5, and ssu72-G33A strains harboring ACT-CUP reporter genes with the CYC1pAmin cleavage/polyadenylation element inserted into the intron in the forward or reverse orientation.

FIG. 5.

Ssu72 mediates both poly(A)-dependent and poly(A)-independent termination. (A) Schematic illustration showing the structure of the tandem G-less cassette constructs used for TRO analysis. Solid rectangles represent sequences derived from the CYC1 3′-flanking region, and the shaded rectangle represents the SNR13 125-232 sequence. (B) Results of G-less cassette TRO analysis of poly(A)-dependent and poly(A)-independent termination in wild-type (WT) and ssu72-G33A mutant strains. Strains were grown at 30°C and then shifted to 37°C for 3 h before the TRO procedure was conducted. Foreach sample, the ratio of total counts incorporated into the distal versus proximal G-less cassettes was determined and was normalized against the ratio for the inert spacer construct in the wild-type strain (CYCds, WT). (C) Bar graph showing averaged results of three experiments like that for which results are shown in panel B. Error bars, standard deviations.