Identification of cis elements directing termination of yeast nonpolyadenylated snoRNA transcripts

Abstract

RNA polymerase II (Pol II) termination is triggered by sequences present in the nascent transcript. Termination of pre-mRNA transcription is coupled to recognition of cis-acting sequences that direct cleavage and polyadenylation of the pre-mRNA. Termination of nonpolyadenylated [non-poly(A)] Pol II transcripts in Saccharomyces cerevisiae requires the RNA-binding proteins Nrd1 and Nab3. We have used a mutational strategy to characterize non-poly(A) termination elements downstream of the SNR13 and SNR47 snoRNA genes. This approach detected two common RNA sequence motifs, GUA[AG] and UCUU. The first motif corresponds to the known Nrd1-binding site, which we have verified here by gel mobility shift assays. We also show that Nab3 protein binds specifically to RNA containing the UCUU motif. Taken together, our data suggest that Nrd1 and Nab3 binding sites play a significant role in defining non-poly(A) terminators. As is the case with poly(A) terminators, there is no strong consensus for non-poly(A) terminators, and the arrangement of Nrd1p and Nab3p binding sites varies considerably. In addition, the organization of these sequences is not strongly conserved among even closely related yeasts. This indicates a large degree of genetic variability. Despite this variability, we were able to use a computational model to show that the binding sites for Nrd1 and Nab3 can identify genes for which transcription termination is mediated by these proteins.

FIG. 1.

Definition of snoRNA 3′ downstream sequences that confer termination function. (A) Northern blot analysis of total RNA derived from a NRD1 WT or a ts nrd1-102 mutant strain at permissive (30°C, 120 min) or restrictive (37°C, 120 min) temperatures. Yeast were transformed with the ADH-snR13(54)-GFP plasmid constructs containing either the 54A or 54B fragment. (B) Northern blot analysis of total RNA derived from NRD1 WT yeast transformed with snR47-GFP plasmids containing various lengths of 3′ downstream sequence. (C) Northern blot analysis of total RNA derived from ts nrd1 or nab3 mutant strains at permissive (30°C, 120 min) or restrictive (37°C, 120 min) temperatures. Yeast were transformed with an ADH-snR47-GFP plasmid containing 116 bp of snR47 3′ downstream sequence. (D) Northern blot analysis of total RNA derived from NRD1 WT yeast transformed with the snR47 15-bp deletion series plasmids. The blot was probed for GFP as well as an SCR1 loading control. The Δ70 construct was set at a value of 1, and all other deletions were expressed as a fraction of this maximum. The snR47(204) plasmid contained an intact snR47 3′ downstream sequence from which all the deletions were derived.

FIG. 2.

Assay for mutant snoRNA termination elements. (A) Growth of yeast harboring the plasmids shown at right. Note the lack of growth on 10 mM 3AT (snR47) and 2.5 mM 3AT (snR13) when snoRNA sequences are present in front of the HIS3 gene. (B) Design of strategy to generate a library of mutant snoRNA termination elements. snR47 70 bp or snR13 66 bp were doped at 1% per base. The doped oligonucleotide was used as an upstream primer to generate a PCR product with ends homologous to a gapped vector. The PCR product and gapped vector were transformed together into yeast. See Materials and Methods for specific protocols.

FIG. 3.

Distribution of mutations in snR13 (A) or snR47 (B) snoRNA termination elements. The mutation frequency at each base is plotted for selected (+3AT) and control (−3AT) populations. GUA[AG] and UCUU motifs are underlined. An asterisk underneath the snR47 sequence denotes an A nucleotide that was changed to a T nucleotide in all clones (see Materials and Methods). The positions of the sequences with respect to the 5′ end of the mature snoRNA are indicated at the beginnings and ends of the sequences.

FIG. 4.

Site-directed mutagenesis of snoRNA-GFP constructs. (A) Sequences of a portion of snR13 or snR47 3′ downstream RNAs. Mutations are identified above the sequences. GUA[AG] and UCUU motifs are highlighted. Bars below the sequences represent mutants identified in the selection experiment (Fig. 3) that greatly impair the elements and result in growth at high concentrations of 3AT. (B) Northern blot analysis of total RNA derived from NRD1-HA WT yeast transformed with single mutant plasmids. The blots were probed for GFP as well as an SCR1 loading control. The snR47-Δ70 and snR13-Δ4 constructs were set at a value of 1, and all other deletions were expressed as a fraction of these maxima. snR13-108 and snR47-204 constructs represent basal levels of GFP expression, and all mutations were made from these plasmids.

FIG. 5.

Gel mobility shift assays of purified Nrd1 and Nab3 proteins. Details of the protein purification, probe sequences, and binding reaction conditions are described in Materials and Methods. Each reaction mixture contained a 2 nM concentration of the labeled RNA probe and, from left to right in each set, 0, 0.156, 0.312, 0.625, 1.25, 2.5, 5.0, or 10 μM purified protein.

FIG. 6.

Gel mobility shift competition assays. Binding conditions were as described in Materials and Methods. Each reaction mixture contained 2 nM labeled probe RNA and 1.25 μM Nab3p (A) or Nrd1p (B) protein. The indicated competitor RNAs were at a final concentration of 500 nM (Nab3) or 50 μM (Nrd1).

FIG. 7.

Alignment of snoRNA downstream sequences. In the top panel, sequences downstream of the SNR13 genes of several yeast species were aligned by using CLUSTAL (37). Indicated species are S. cerevisiae (cer), S. mikitae (mik), and S. bayanus (bay). The second panel shows a similar alignment of the SNR47 downstream sequences. In the bottom panel, the downstream regions of several S. cerevisiae snoRNA genes are aligned by their first downstream Nrd1 or Nab3 element to show the presence of sequence elements similar to those present in SNR13 and SNR47.

FIG. 8.

Tests of models predicting non-poly(A) terminators in the S. cerevisiae genome. (A) Receiver operator characteristic (ROC) curves (15) for the training set compared to those for all other open reading frames, snRNAs, and snoRNAs, using the optimized binding models for Nrd1 alone (dashed), Nab3 alone (dotted), and cooperative Nrd1-Nab3 binding (solid). (B) ROC curves for test set RNA genes compared to those for the nontest set. The models and parameters are the same for the training set curves and the test set curves. The ROC curve for a random regulatory model that has no predictive value at all would run along the diagonal, while a ROC curve for a perfectly predictive regulatory model would run up the y axis and then along the top of the plot. (C) Sequence logo representations (31) of the binding specificities of Nrd1 and Nab3 optimized for the cooperative model, Nrd1 alone, and Nab3 alone. The y axis shows bits of information. The line patterns of the boxes surrounding the sequence logos correspond to the lines in the ROC curves.