Identification of a role for histone H2B ubiquitylation in noncoding RNA 3'-end formation through mutational analysis of Rtf1 in Saccharomyces cerevisiae

Abstract

The conserved eukaryotic Paf1 complex regulates RNA synthesis by RNA polymerase II at multiple levels, including transcript elongation, transcript termination, and chromatin modifications. To better understand the contributions of the Paf1 complex to transcriptional regulation, we generated mutations that alter conserved residues within the Rtf1 subunit of the Saccharomyces cerevisiae Paf1 complex. Importantly, single amino acid substitutions within a region of Rtf1 that is conserved from yeast to humans, which we termed the histone modification domain, resulted in the loss of histone H2B ubiquitylation and impaired histone H3 methylation. Phenotypic analysis of these mutations revealed additional defects in telomeric silencing, transcription elongation, and prevention of cryptic initiation. We also demonstrated that amino acid substitutions within the Rtf1 histone modification domain disrupt 3'-end formation of snoRNA transcripts and identify a previously uncharacterized regulatory role for the histone H2B K123 ubiquitylation mark in this process. Cumulatively, our results reveal functionally important residues in Rtf1, better define the roles of Rtf1 in transcription and histone modification, and provide strong genetic support for the participation of histone modification marks in the termination of noncoding RNAs.

Figure 1.—

Isolation of mutations that alter the Rtf1 HMD. (A) Alignment of Rtf1 protein sequences from a range of eukaryotic species generated using the Clustal X algorithm (Larkin et al. 2007). Notations above the amino acids indicate the degree of conservation with * denoting invariant residues. S. cerevisiae Rtf1 mutants were generated through targeted substitutions (underlined) and unbiased genetic screening (arrows). The F80V substitution (open arrowhead) was obtained in combination with F123S. Subcloning of these mutations revealed that F123S is primarily responsible for the phenotypes of this double mutant. (B) Dilution growth assays of HA-RTF1 RKR1 (KY2076), HA-RTF1 rkr1∆ (KY2075), HA-rtf1∆3 (KY2032), rkr1∆ HA-rtf1∆3 (KY2029), HA-rtf1∆4 (KY2033), rkr1∆ HA-rtf1∆4 (KY2030), HA-rtf1-F123S (KY1531), rkr1∆ HA-rtf1-F123S (KY1957), HA-rtf1-F80V,F123S (KY2034), rkr1∆ HA-rtf1F80V,F123S (KY2031), HA-rtf1-E104K (KY1532), rkr1∆ HA-rtf1-E104K (KY1961), HA-rtf1-E104G (KY1533), and rkr1∆ HA-rtf1-E104G (KY1964) cells. Ten-fold serial dilutions were spotted on SD complete (left) and SC complete (right) media and incubated at 30° for 3 days.

Figure 2.—

Amino acid substitutions in the Rtf1 HMD cause phenotypes associated with defects in transcription. (A) Ten-fold serial dilutions of RTF1 (KY1010), rtf1Δ (KY619), HA-rtf1Δ3 (KY1506), HA-rtf1Δ4 (KY1508), HA-rtf1-102-104A (KY1510), and HA-rtf1-108-110A (KY1511) cells were spotted on SC −Ura medium containing 50 μg/ml 6-AU to assess 6-AU sensitivity (left), SD −His medium to examine the Spt⁻ phenotype (middle), or SC complete medium (right) as a control for growth. (B) Ten-fold serial dilutions of RTF1 (KY1021), rtf1Δ (KY656), HA-rtf1Δ3 (KY1507), HA-rtf1-E104K (KY1532), HA-rtf1-E104G (KY1533), HA-rtf1Δ4 (KY1508), HA-rtf1-F80V-F123S (KY1512), and HA-rtf1-F123S (KY1531) cells were spotted on SC −Ura medium containing 50 μg/ml 6-AU to assess 6-AU sensitivity (left) or SC −Ura medium as a control for growth (right). (C) Ten-fold serial dilutions of RTF1 (KY292), rtf1Δ (KY522), HA-rtf1Δ3 (KY1507), HA-rtf1-E104K (KY1514), HA-rtf1-E104G (KY1515), HA-rtf1Δ4 (KY1508), HA-rtf1-F80V,F123S (KY1512), and HA-rtf1-F123S (KY1513) cells were spotted on SD −His medium to examine the Spt⁻ phenotype (left) or SD complete medium (right) as a control for growth. Plates were incubated at 30° for 4 days.

Figure 3.—

Amino acid substitutions in the Rtf1 HMD cause phenotypes related to defects in chromatin structure. (A) To analyze telomeric silencing in strains expressing Rtf1 substitution mutants, 10-fold serial dilutions of RTF1 (KY1226), rtf1Δ (KY1227), HA-rtf1Δ3 (KY2035), HA-rtf1Δ4 (KY2036), HA-rtf1-102-104A (KY2037), and HA-rtf1-108-110A (KY2038) cells were spotted on SC −Ura medium containing 5-FOA to assess telomeric silencing defects (left) or SC complete medium (right) as a control for growth. Plates were incubated at 30° for 3 days. (B) To measure aberrant initiation from a cryptic promoter within the FLO8 open reading frame, 10-fold serial dilutions of RTF1 (KY1116), rtf1Δ (KY1207), HA-rtf1Δ3 (KY1208), HA-rtf1Δ4 (KY1209), HA-rtf1-102-104A (KY1210), and HA-rtf1-108-110A (KY1211) cells were spotted on SC −His +galactose medium to induce and monitor expression of the GAL1pr-FLO8-HIS3 reporter (left) and SC complete medium (right) as a control for growth. Plates were incubated at 30° for 7 days. (C) Ten-fold serial dilutions of RTF1 (KY1491), rtf1Δ (KY1370), HA-rtf1Δ3 (KY1230), HA-rtf1-E104K (KY1526), HA-rtf1-E104G (KY1527), HA-rtf1Δ4 (KY1231), HA-rtf1 F80V-F123S (KY1234), and HA-rtf1-F123S (KY1525) were spotted on SC −Ura medium containing 5-FOA to assess telomeric silencing defects (left), SC −His +galactose medium to induce and monitor expression of the GAL1pr-FLO8-HIS3 reporter (middle), and SC complete medium as a control for growth (right). Plates were incubated at 30° for 6 days.

Figure 4.—

Immunoblot analysis reveals amino acids in Rtf1 important for histone H3 methylation. Immunoblot analysis of Rtf1-dependent histone H3 modifications was performed on strains containing Rtf1 internal deletions and amino acid substitutions. Whole cell extracts from RTF1 (KY1491), HA-RTF1 (KY2088), rtf1Δ (KY1370), HA-rtf1Δ3 (KY1230), HA-rtf1Δ4 (KY1231), HA-rtf1-102-104A (KY1232), HA-rtf1-108-110A (KY1233), HA-rtf1-F123S (KY1525), HA-rtf1-E104K (KY1526), HA-rtf1-E104G (KY1527), set1∆ (KY907), and dot1∆ (KY903) strains were probed with antibodies specific for histone H3 K4me3, H3 K4me2, H3 K79me2/3 (both methylation states are detected by this antibody), and total histone H3 protein. A nonspecific band detected with the H3 K79me2/3 antibody is noted by an asterisk. Immunoblots were probed with antibodies specific for the HA epitope tag to confirm Rtf1 expression and for G6PDH to serve as a loading control. Note that Rtf1 is sensitive to proteolytic attack under certain extraction methods, such as that used in this experiment, and occasionally runs as two bands.

Figure 5.—

Histone H2B K123 ubiquitylation requires E104 and F123 residues within Rtf1. Immunoblot analysis of histone H2B K123 ubiquitylation in strains expressing alanine scanning mutations (A) or screen-derived point mutations (B). For A, the following strains were used: HA-RTF1 (KY1459), rtf1Δ (KY982), HA-rtf1-102-104A (KY2039), and HA-rtf1-108-110A (KY2040). For B, the following strains were used: RTF1 (KY943), HA-RTF1 (KY1459), rtf1Δ (KY982), HA-rtf1Δ3 (KY1216), HA-rtf1Δ4 (KY1217), HA-rtf1-F80V-F123S (KY1462), HA-rtf1-F123S (KY1534), HA-rtf1-E104K (KY1535), and HA-rtf1-E104G (KY1536). Cells were transformed with plasmids expressing either untagged or FLAG-tagged histone H2B, and extracts from these strains were probed with an antibody specific for the FLAG epitope. The bands corresponding to unmodified H2B (FLAG-H2B) and ubiquitylated H2B (FLAG-H2Bub) are indicated. Extracts were probed with antibodies specific for the HA epitope to confirm Rtf1 expression and for G6PDH, which serves as a loading control.

Figure 6.—

Amino acid substitutions in Rtf1 impair transcription termination in a SNR47 reporter plasmid. (A) rtf1∆ cells (KY2041) transformed with either pADH1-SNR47(70)-HIS3-CYC1 (left) or pADH1-HIS3-CYC1 (right) were subsequently transformed with pRS314 or pRS314-derived plasmids containing HA-tagged RTF1 (pLS21-5), rtf1-102-104A (pJB1), rtf1-E104K (pCD1), rtf1-108-110A (pJB2), or rtf1-F80V,F123S (pKB1) as indicated. Ten-fold serial dilutions of these cells were spotted on SC −Leu −Trp −His and SC −Leu −Trp media and incubated for 3 days at 30°. (B) Northern blot analysis was used to examine HIS3 transcripts. Analysis was done using 20 μg of total RNA from the following his3∆200 strains: RTF1 (KY2090), RTF1 (KY2042) transformed with pADH1-HIS3-CYC1, rtf1∆ (KY2041) containing pADH1-SNR47(70)-HIS3-CYC1 subsequently transformed with pRS314 or pRS314-derived plasmids containing HA-tagged RTF1 (pLS21-5), rtf1-102-104A (pJB1), or rtf1-E104K (pCD1) as indicated by listed genotype.

Figure 7.—

Amino acid substitutions in Rtf1 prevent proper snoRNA 3′-end formation. (A) Representative Northern analysis of extended SNR47 transcripts (SNR47-YDR042C) in wild-type (KY1698), rtf1∆ (KY1703), HA-rtf1-102-104A (KY1981), HA-rtf1-E104K (KY1982), HA-rtf1-108-110A (KY1983), and HA-rtf1-F80V,F123S (KY1984) cells. SCR1 serves as a loading control for all Northern analyses. Twenty micrograms of total RNA was analyzed in these experiments. (B) Quantification of SNR47-YDR042C transcript levels from three independent Northern analyses, performed as described in A, with relative signal of wild type set to one as described in materials and methods. (C) Representative Northern analysis of extended SNR13 transcripts (SNR13-TRS31) and TRS31 transcripts in wild-type (KY1698), rtf1∆ (KY1703), HA-rtf1-102-104A (KY1981), HA-rtf1-E104K (KY1982), HA-rtf1-108-110A (KY1983), and HA-rtf1-F80V,F123S (KY1984) cells. Twenty-five micrograms of total RNA was analyzed in these experiments. (D) Quantification of SNR13-TRS31 transcript levels from three independent Northern analyses, performed as described in C, with relative signal of wild type set to one.

Figure 8.—

Identification of a role for H2B K123 ubiquitylation in promoting 3′-end formation of snoRNA transcripts. (A) rtf1∆ (KY2041), wild-type (KY2042), hta2∆ htb2∆ (KY2043), htb1-K123R hta2∆ htb2∆ (KY2044), rad6∆ (KY2045), bre1∆ (KY2046), and rad6∆ bre1∆ (KY2047) cells were transformed with either pADH1-SNR47(70)-HIS3-CYC1 (left) or pADH1-HIS3-CYC1 (right). Ten-fold serial dilutions of these cells were spotted on SC −Leu −His and SC −Leu medium and incubated for 4 days at 30°. (B) Representative Northern analysis of extended SNR47 transcripts (SNR47-YDR042C) in hta2∆ htb2∆ (KY2043) and htb1-K123R hta2∆ htb2∆ (KY2044) cells. Twenty micrograms of total RNA was analyzed. SCR1 served as a loading control for all Northern analyses. (C) Quantification of SNR47-YDR042C transcript levels from three independent Northern analyses, performed as in B, with relative signal of hta2∆ htb2∆ set to one as described in materials and methods. (D) Representative Northern analysis of extended SNR47 transcripts (SNR47-YDR042C) in wild-type (KY2048), rad6∆ (KY2045), and bre1∆ (KY2046) cells. Twenty-five micrograms of total RNA was analyzed. (E) Quantification of SNR47-YDR042C transcript levels from three independent Northern analyses, performed as in D, with relative signal of wild type set to one. (F) Representative Northern analysis of extended SNR13 transcripts (SNR13-TRS31) in wild-type (KY2042), hta2∆ htb2∆ (KY2043), htb1-K123R hta2∆ htb2∆ (KY2044), rad6∆ (KY2045), and bre1∆ (KY2046) cells. Twenty-five micrograms of total RNA was analyzed. (G) Quantification of SNR13-TRS31 transcript levels from three independent Northern analyses performed as described in F. The relative signals of hta2∆ htb2∆ (left graph) or wild type (right graph) were set to one. (H) Wild-type (KY2042), dot1∆ (KY2091), set1∆ (KY2092), set1∆ dot1∆ (KY2093), rtf1∆ (KY2041), bre1∆ (KY2046), rad6∆ (KY2045), and rad6∆ bre1∆ (KY2047) cells were transformed with either pADH1-SNR47(70)-HIS3-CYC1 (left) or pADH1-HIS3-CYC1 (right). Ten-fold serial dilutions of these cells were spotted on SC −Leu −His and SC −Leu medium and incubated for 4 days at 30°.