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Yeast arginine methyltransferase Hmt1p regulates transcription elongation and termination by methylating Npl3p - PubMed

Yeast arginine methyltransferase Hmt1p regulates transcription elongation and termination by methylating Npl3p

Chi-Ming Wong et al. Nucleic Acids Res. 2010 Apr.

Abstract

The heterogeneous nuclear ribonucleoprotein Npl3p of budding yeast is a substrate of arginine methyltransferase Hmt1p, but the role of Hmt1p in regulating Npl3p's functions in transcription antitermination and elongation were unknown. We found that mutants lacking Hmt1p methyltransferase activity exhibit reduced recruitment of Npl3p, but elevated recruitment of a component of mRNA cleavage/termination factor CFI, to the activated GAL10-GAL7 locus. Consistent with this, hmt1 mutants displayed increased termination at the defective gal10-Delta56 terminator. Remarkably, hmt1Delta cells also exhibit diminished recruitment of elongation factor Tho2p and a reduced rate of transcription elongation in vivo. Importantly, the defects in Npl3p and Tho2p recruitment, antitermination and elongation in hmt1Delta cells all were mitigated by substitutions in Npl3p RGG repeats that functionally mimic arginine methylation by Hmt1p. Thus, Hmt1p promotes elongation and suppresses termination at cryptic terminators by methylating RGG repeats in Npl3p. As Hmt1p stimulates dissociation of Tho2p from an Npl3p-mRNP complex, it could act to recycle these elongation and antitermination factors back to sites of ongoing transcription.

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Figures

Figure 1.
Figure 1.

Hmt1p reduces termination at the defective gal10-Δ56 terminator. (A) Diagram summarizing results in (B) and (C) indicating that hmt1Δ increases termination at the gal10-Δ56 terminator, reducing the RT transcript while increasing GAL7 transcript abundance, thereby suppressing the Gal− phenotype in gal10-Δ56 cells. (B) Serially diluted strains of the indicated genotypes were spotted on synthetic complete medium lacking leucine (SC-LEU) plates containing 2% glucose (left) or 2% galactose (right) and incubated for 4 days. (C) Upper panel: northern blot analysis of GAL7 and RT transcripts. Total RNA (20 µg) from indicated strains induced with 2% galactose for 3 h were resolved and probed for GAL7 mRNA. The GAL7/RT ratios were determined by densitometry. The values indicated are the means and standard deviations of ratios calculated from three replicate experiments, which gave highly similar results. Lower panel: ethidium bromide staining of the gel.

Figure 2.
Figure 2.

hmt1Δ reduces the rate of transcription elongation in vivo. (A) Diagram of the PGAL1-YLR454w gene with positions of PCR primers indicated (in bp) from nucleotide A of the start codon. (B, C) Quantification of Rpb3p occupancy measured by ChIP with antiRpb3p antibodies at the five positions across PGAL1-YLR454w indicated in (A) for HMT1 (CMY125) (B) or hmt1Δ (CMY130) (C). Cells grown in YP containing galactose were treated with 4% glucose for the indicated times. DNA extracted from immunoprecipitates and input chromatin samples was amplified by PCR in the presence of [33P]dATP for the different regions of PGAL1-YLR454w, together with a fragment from a non-transcribed region of Chr I to control for non-specific immunoprecipitation. PCR products were quantified with by phosphorimaging and ratios of experimental-to-control signals in the immunoprecipitates were normalized for the corresponding ratios for input samples to yield the occupancy values. Occupancies were normalized to those derived from cells grown in 2% galactose for 2 h (time = 0). (D) hmt1Δ does not detectably affect RNAP II processivity in vivo. HMT1 and hmt1Δ cells were grown in galactose medium (time = 0) and quantification of Rpb3p occupancy was performed as above. The value at each position was normalized to that of the WT strain. (E−G) Methyltransferase activity of Hmt1p stimulates the rate of transcription elongation in vivo. hmt1Δ cells containing empty vector (E) or plasmids harboring hmt1-G68R (F) or HMT1 (G) were induced with SC-LEU 2% galactose for 2 h (time = 0) and treated with 4% glucose for the indicated times. ChIP analysis of Rpb3p across the PGAL1-YLR454w gene was conducted as described earlier. Average results obtained from two independent cultures and two PCR amplifications for each culture were plotted in the histograms.

Figure 3.
Figure 3.

Loss of HMT1 affects Npl3p and Hrp1p occupancies at activated GAL10-GAL7. (A) Diagram of the GAL10-GAL7 locus, with PCR primer pairs indicated with black bars and terminator labeled as ‘T’. (B) ChIP analysis of Npl3p (upper panel) and Hrp1p (middle panel) occupancies across the GAL10-GAL7 locus. ChIP analysis of Myc-Npl3p or Myc-Hrp1p occupancies was conducted using antimyc antibodies as described in Figure 2, except using primers to amplify different regions of GAL10-GAL7 in panel A and to amplify non-transcribed region of Chr V was conducted to control for non-specific immunoprecipitation. Upper panel, transformants of hmt1Δ NPL3-myc strain (CMY201) containing empty vector (Lanes 1–5), or plasmids harboring hmt1-G68A (Lanes 6–10), hmt1-G68R (Lanes 11–15) or HMT1 (Lanes 16–20) were grown in SC-LEU galactose medium. Middle panel, transformants of hmt1Δ HRP1-myc strain (CMY203) harboring the same plasmids described for the upper panel were analyzed as described earlier. Lower panel: Analysis of input chromatin samples for the CMY201 transformants. (C) Quantification of Npl3p occupancy measured at the indicated positions. The ratios of experimental-to-control signals in the immunoprecipitate samples (B, upper panel) were normalized for the corresponding ratios for input samples (B, lower panel) to yield the occupancy values. Average results obtained from two independent cultures and two PCR amplifications for each culture were plotted. (D) Possible effect of mutation or deletion of HMT1 on expression of Myc-tagged Npl3p was analyzed by western blot analysis of WCEs of the relevant strains with antiMyc and antiPgk1p antibodies (to provide a loading control). (E) Quantification of Hrp1p occupancy measured at the indicated positions. The ratios of experimental to-control signals in the immunoprecipitate samples (B, middle panel) were normalized for the corresponding ratios for input samples (B, lower panel) to yield the occupancy values. Average results obtained from two independent cultures and two PCR amplifications for each culture were plotted. (F) Possible effect of mutation or deletion of HMT1 on expression of Myc-tagged Hrp1p was analyzed by western blot analysis of WCEs of the relevant strains with antiMyc and antiPgk1p antibodies (to provide a loading control).

Figure 4.
Figure 4.

Npl3RKp suppresses the antitermination and elongation rate defects caused by deletion of NPL3 or HMT1. (A) ChIP analysis of Npl3p or Npl3RKp in HMT1 or hmt1Δ strains. HMT1 NPL3-myc (YAM535, Lanes 1−5), hmt1ΔNPL3-myc (YAM533, Lanes 6−10) and hmt1Δnpl3RK-myc (YAM534, Lanes 11−15) were analyzed and the Npl3p or Npl3RKp occupancies across the GAL10-GAL7 locus were determined as described in Figure 3. Average results obtained from two independent cultures and two PCR amplifications for each culture were plotted. (B) Npl3RKp suppresses the Gal+ phenotype (antitermination defect) of npl3Δgal10-Δ56 (CMY129) cells. npl3Δgal10-Δ56 cells transformed with empty vector, NPL3 or npl3RK plasmid were spotted on YP plates containing either 2% glucose (Glu, left panel) or 2% galactose (Gal, right panels) as carbon source and incubated for 3 days in YP. Note that YP was used instead of SC-LEU medium because the extreme slow-growth phenotype conferred by npl3Δon SC-LEU+Gal medium, evident in GAL10 cells, obscures suppression of the Gal− phenotype of gal10-Δ56 by npl3Δ(data not shown). (C) Northern blot analysis of GAL7 and GAL10-GAL7 RT mRNAs conducted as in Figure 1C. The values indicated are the means and standard deviations of ratios calculated from three replicate experiments, which gave highly similar results. (D) Npl3RKp suppresses the Gal+ phenotype (antitermination defect) of hmt1Δgal10-Δ56 (CMY223) cells. HMT1 gal10-Δ56 and hmt1Δgal10-Δ56 strains with plasmid-borne NPL3 or npl3RK were spotted on SC-LEU plates containing 2% glucose (left) or 2% galactose (right) and incubated for 4 days. (E) ChIP analysis of Rpb3p occupancy across the PGAL1-YLR454w locus after imposing glucose repression. hmt1Δ cells (CMY130) with or without plasmid-borne npl3RK were induced with 2% galactose for 2 h (time = 0) and treated with 4% glucose for the indicated times. ChIP assays were done as described in Figure 2. PCR amplifications of input chromatin samples are labeled ‘In’; non-coding region on chromosome I (Chr I) was amplified as a control for non-specific immunoprecipitation. (F, G) Quantification of Rpb3p occupancies measured in (E) at the indicated positions at PGAL1-YLR454w in hmt1Δ cells (F) and hmt1Δ cells containing npl3RK (G). Average results obtained from two independent cultures and two PCR amplifications for each culture were plotted. (H) Possible effect of hmt1Δ on Myc-tagged Npl3p and Myc-tagged Np3RKp levels was examined by western blot analysis of WCEs from the relevant strains with antiMyc and antiPgk1p antibodies.

Figure 5.
Figure 5.

Tho2p recruitment and gene length-dependent transcription defects in hmt1Δ cells are suppressed by npl3RK, and a hypothetical model for the role of Hmt1p in recycling Tho2p and Npl3p from mRNPs to sites of transcription. ChIP analysis of Tho2p occupancy at the 5′ region of GAL10 gene (region B in Fig. 3A) in hmt1Δ THO2-myc13 (CMY202) cells. Cells transformed with empty vector, or plasmids containing hmt1-G68A, hmt1-G68R, HMT1, NPL3 or npl3RK were grown in SC-LEU with 2% galactose and ChIP analysis was conducted using antiMyc antibodies (to measure Tho2p occupancy) or antibodies against Rpb3p (to measure RNAP II occupancy). Non-coding region on chromosome V (Chr V) was amplified as control for non-specific immunoprecipitation. The occupancies were quantified and plotted for Tho2p (A) and Rpb3p (B). Average results obtained from two independent cultures and two PCR amplifications for each culture were plotted. The occupancies of Tho2p at the GAL10 ORF are significantly lower in control (hmt1Δ), G68A and G68R versus HMT1 cells, and in cells containing extra copies of NPL3 versus extra copies of npl3RK, as judged by the Student’s t-test, with P < 0.01(*). (C) HMT1 (BY4741) and hmt1Δ (3171) cells transformed with reporter plasmid pSCH209-LAC4 plus plasmids harboring hmt1-G68A, NPL3, npl3RK or empty vector were grown in SC-LEU-URA with 2% galactose. HMT1 (BY4741) and hmt1Δ (3171) cells harboring pSCH202 were analyzed as controls. Acid phosphatase activity was measured in cell lysates and the GLAM ratio was calculated as the ratio of activities in transformants harboring pSCH209-LAC4 versus those containing pSCH202. Results are the averages from three independent experiments and error bars indicate SD (D) Model depicting how Hmt1p regulates transcription elongation and termination by methylating Npl3p. Upper panel: In WT cells, methylation of Npl3p by Hmt1p allow dissociation the TREX(Tho2p)−Npl3p−mRNP to facilitate (1) TREX(Tho2p) recruitment to sites of transcription and (2) nuclear export of the Npl3p−mRNP. Subsequent nuclear import and methylation of Npl3p by Hmt1p enables its recruitment to sites of transcription (3). Npl3p and TREX both stimulate elongation and, by impeding recruitment of Hrp1p (4), Npl3p prevents transcription termination at cryptic terminators. Middle panel, in hmt1Δ NPL3 cells, the failure to methylate Npl3p causes Tho2p (TREX) and Npl3p to be sequestered in mRNPs that are not exported efficiently; the ensuing impaired recruitment of TREX(Tho2p) and Npl3p to sites of transcription reduces the rate of elongation, and the resulting increased Hrp1p occupancy enables transcription termination at cryptic terminators. Lower panel, in hmt1Δ npl3RK cells, TREX(Tho2p)-Npl3RKp-mRNP can dissociate independently of Npl3p methylation to recycle TREX(Tho2p) and Npl3RKp back to sites of transcription where they stimulate elongation and block termination at cryptic terminators, as occurs in WT cells.

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