RNA polymerase II elongation factors Spt4p and Spt5p play roles in transcription elongation by RNA polymerase I and rRNA processing - PubMed (original) (raw)

RNA polymerase II elongation factors Spt4p and Spt5p play roles in transcription elongation by RNA polymerase I and rRNA processing

D A Schneider et al. Proc Natl Acad Sci U S A. 2006.

Abstract

Previous investigations into the mechanisms that control RNA Polymerase (Pol) I transcription have primarily focused on the process of transcription initiation, thus little is known regarding postinitiation steps in the transcription cycle. Spt4p and Spt5p are conserved throughout eukaryotes, and they affect elongation by Pol II. We have found that these two proteins copurify with Pol I and associate with the rDNA in vivo. Disruption of the gene for Spt4p resulted in a modest decrease in growth and rRNA synthesis rates at the permissive temperature, 30 degrees C. Furthermore, biochemical and EM analyses showed clear defects in rRNA processing. These data suggest that Spt4p, Spt5p, and, potentially, other regulators of Pol I transcription elongation play important roles in coupling rRNA transcription to its processing and ribosome assembly.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.

Fig. 1.

Spt4/5 associates with Pol I. Pol I was immunoprecipitated from equivalent amounts of crude extracts made from NOY761 (HA-Rrn3p) and NOY2164 (HA-Spt5p) by using a polyclonal antibody against A190. For control, identical extracts were treated with protein A-Sepharose beads only. Coprecipitated HA-tagged Rrn3p and Spt5p were detected by Western blot using the monoclonal antibody 12CA5.

Fig. 2.

Fig. 2.

Spt4p and Spt5p associate with rDNA in vivo. ChIP analysis of (His)7-(HA)3-tagged Spt4p, Spt5p, and A135 (Pol I) was performed by using strains NOY2165, NOY2164, and NOY2166, respectively. NOY388 was also analyzed as “no tag” control. Anti-HA antibody 12CA5 was used for IP, and two dilutions of both input and IP DNA were analyzed by PCR to verify that the data were in the linear range of detection. Two regions of the rDNA were examined: the promoter region from −251 to −24 and the 25S coding region from +3586 to +3876. Numbering is relative to the initiation site of Pol I. ChIP data from three independent cultures were quantified by using a PhosporImager, and the ratios of IP/input were averaged for each region of the rDNA and were normalized to the values for Pol I.

Fig. 3.

Fig. 3.

Processivity analysis of Pol I transcription in the _spt4_Δ strain relative to WT. NOY388 (WT) and NOY2167 (_spt4_Δ) (both carrying pRS316) were grown to A600 = ≈0.3 in SD-Ura medium. (A) Pol I association with the 5′ end (+67 to +388) and the 3′ end (+6067 to +6388) of the 35S rRNA coding region was then measured by ChIP using anti-A190 antibodies. Two dilutions of input and IP DNA were measured by PCR. As a negative control, duplicate samples were treated with protein A-Sepharose beads only (“−Ab”) and diluted similarly to IP samples. (B) For each strain, the IP/input value for the 3′ end was divided by the value for the 5′ end, and the ratio (“processivity”) for the mutant was normalized to that for WT. The values obtained from four independent IPs from two independent cultures were averaged. Raw values for IP/input were 0.060 ± 0.014 for the 5′ end of WT, 0.013 ± 0.002 for the 3′ end of WT, 0.124 ± 0.004 for the 5′ end of _spt4_Δ, and 0.017 ± 0.003 for the 3′ end of _spt4_Δ.

Fig. 4.

Fig. 4.

Representative EMs of rRNA genes from the _spt4_Δ strain relative to WT. (A) NOY388 (WT) and NOY2167 (_spt4_Δ) were grown in YEPD plus 1 M sorbitol to midlog phase. Miller spreads were analyzed by EM, and two active genes are shown for each strain. (Scale bars, 0.5 μm.) (B) The number of Pol I molecules per gene was counted, and distribution of Pol I among genes is plotted by grouping with an increment of 5. (C) Numerical values for the data presented in B and the active rDNA repeats (as percentage of total) counted for contiguously traced rDNA regions (23) are shown. The values for the mutant normalized to those for the WT (100) are also shown in parentheses. N, the number of genes examined per strain; pols, polymerases.

Fig. 5.

Fig. 5.

rDNA copy number is reduced in the _spt4_Δ strain compared with WT. (A) genomic DNA was extracted from three independent isolates of _spt4_Δ (NOY2167) and three independent WT cultures (NOY388) as well as from cultures with known rDNA copy numbers: NOY1071 (25 copies), NOY886 (42 copies), and NOY1064 (143 copies). After digestion with HindIII, DNA was separated in a 1.5% agarose gel and transferred to a nylon membrane. Southern hybridization was performed using 32P-labeled DNA probes against rDNA and the single copy control gene URA3. To visualize the URA3 probe clearly (Lower), exposure time was extended. (B) Data from A were quantified by using a PhosphorImager, and the ratio of rDNA/URA3 of the reference strains was plotted against copy number. From the resulting linear regression, the average copy number in WT and _spt4_Δ was calculated. Variations (standard deviations) among three independent cultures were ≈15% for both WT and _spt4_Δ.

Fig. 6.

Fig. 6.

Processing of rRNA transcripts is impaired in the spt4_Δ strain. (A) One of the two genes shown in Fig. 4_A for NOY388 (WT) and for NOY2167 (_spt4_Δ) is shown in higher magnification with notes indicating the absence of cotranscriptional cleavage of the 35S rRNA in the gene from the _spt4_Δ strain. (Scale bars, 0.5 μm.) (B) Quantification of SSU processome formation and cotranscriptional cleavage of rRNA observed by EM in the WT and _spt4_Δ strains, classified as efficient, intermediate, or slow (26). (C) The two strains were grown in SD (−Met) media and were pulse-labeled for 2.5 and 5 min with [methyl-3H]methionine. RNA was isolated and analyzed by using formaldehyde-agarose gel electrophoresis, followed by autoradiography. Positions of rRNA species are indicated. Two exposures of the blot are shown [1 day (Left) and 1 week (Right)]. The ratios of precursors to mature products detected after 2.5-min pulse-labeling are indicated, normalized to WT. (D) Analysis of polysomes and ribosomal subunits in the two strains. Cells were grown in YEPD medium at 30°C, and extracts were analyzed by sucrose density gradient centrifugation. Positions of 40S, 60S, and 80S as well as polysomes are indicated. The arrows with an H indicate halfmers in the _spt4_Δ strain.

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References

    1. Grummt I. Genes Dev. 2003;17:1691–1702. - PubMed
    1. Nomura M. Cold Spring Harbor Symp. Quant. Biol. 2001;66:555–565. - PubMed
    1. Nomura M., Nogi Y., Oakes M. In: The Nucleolus, Olson M. O. J., editor. Austin, TX: Landes; 2004. pp. 128–153.
    1. Cavanaugh A., Hirschler-Laszkiewicz I., Rothblum L. In: The Nucleolus, Olson M. O. J., editor. Georgetown, TX: Landes Bioscience; 2004. pp. 88–127.
    1. Russell J., Zomerdijk J. C. Trends Biochem. Sci. 2005;30:87–96. - PMC - PubMed

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