RNA polymerase I transcription and pre-rRNA processing are linked by specific SSU processome components - PubMed (original) (raw)

Comparative Study

. 2004 Oct 15;18(20):2506-17.

doi: 10.1101/gad.1226604.

Affiliations

Comparative Study

RNA polymerase I transcription and pre-rRNA processing are linked by specific SSU processome components

Jennifer E G Gallagher et al. Genes Dev. 2004.

Abstract

Sequential events in macromolecular biosynthesis are often elegantly coordinated. The small ribosomal subunit (SSU) processome is a large ribonucleoprotein (RNP) required for processing of precursors to the small subunit RNA, the 18S, of the ribosome. We have found that a subcomplex of SSU processome proteins, the t-Utps, is also required for optimal rRNA transcription in vivo in the yeast Saccharomyces cerevisiae. The t-Utps are ribosomal chromatin (r-chromatin)-associated, and they exist in a complex in the absence of the U3 snoRNA. Transcription is required neither for the formation of the subcomplex nor for its r-chromatin association. The t-Utps are associated with the pre-18S rRNAs independent of the presence of the U3 snoRNA. This association may thus represent an early step in the formation of the SSU processome. Our results indicate that rRNA transcription and pre-rRNA processing are coordinated via specific components of the SSU processome.

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Figures

Figure 1.

Figure 1.

Pre-rRNA processing in S. cerevisiae. In yeast the rDNA is found in a 9.1-kb unit repeated 100–200 times. The 35S pre-rRNA is transcribed by RNA polymerase I (Pol I). In some instances the 35S is cleaved at A3, yielding the 23S pre-rRNA, which is then processed at A0, A1, and A2 to yield the 20S precursor. In other instances, the 35S pre-rRNA is processed sequentially at A0, A1, and A2 to yield the 20S precursor. The 20S precursor is exported to the cytoplasm and matured into the 18S rRNA as part of the small ribosomal subunit (SSU). The 27SA3 pre-rRNA is then processed to generate the 5.8S and 25S rRNAs of the large ribosomal subunit (LSU). The 27SA2 precursor is processed into the 5.8S and 25S rRNAs of the LSU. The U3 snoRNA-dependent cleavages are in bold. The location of oligonucleotides b, c, and e used to probe for pre-rRNAs on Northern blots is indicated.

Figure 2.

Figure 2.

Depletion of different components of the SSU processome leads to varying affects on steady-state pre-rRNA levels. Seventeen strains were constructed in which each Utp was expressed from a conditional (GAL) promoter (GAL::HA-UTP; Dragon et al. 2002). RNA was harvested from strains either prior to (undepleted [U]) or after (depleted [D]) depletion, as by Dragon et al. (2002). Following gel electrophoresis, the RNA was analyzed by Northern blots with specific oligonucleotides. The boxed names indicate the subgroup of Utps whose depletion affects levels of all pre-rRNAs (t-Utps). (A) Northern blots were probed with oligo c (between A2 and A3; Fig. 1). The expected precursors are labeled. (B) Northern blots were probed with oligo b (between A1 and A2; Fig. 1) and oligo e (in ITS2; Fig. 1).

Figure 3.

Figure 3.

Transcription run-on analysis indicates that depletion of a subgroup of SSU processome components leads to a reduction in pre-rRNA transcription. Each strain carries the single gene encoding the indicated protein under the control of a GAL promoter (GAL::HA-UTP), allowing for conditional expression. The boxes indicate proteins whose depletion affects levels of all pre-rRNAs (from Fig. 2), and that were therefore hypothesized to be affecting transcription. The NOY504 strain carries a temperature-sensitive RNA polymerase I. (A) The Utp proteins are depleted when the GAL::HA-UTP strains are grown in glucose. Western blots with anti-HA antibody were performed on protein harvested from yeast expressing the indicated tagged proteins under the control of a GAL promoter prior to (0) or after (3, 6, 9, 24 h) the switch to glucose. (B) Growth curves of yeast strains. Growth of the GAL::HA-UTP and the parent YPH499 strains in glucose is indicated graphically and compared to growth of the NOY504 strain at the permissive (room temperature [RT]) and nonpermissive (37°C) temperatures. (C) The rDNA transcription unit. RNA polymerase I transcribes the 35S primary rRNA transcript, whereas RNA polymerase III transcribes the 5S rRNA in the opposite direction. PCR products corresponding to the indicated segments of the nontranscribed spacer (NTS), the 5′ external transcribed spacer (5′ETS), and the 25S rRNA were cloned into the pTOPO plasmid. (D) Transcription run-on analysis. The GAL::HA-UTP strains were either not depleted or depleted for 6 h of the indicated proteins, permeabilized, and exposed to α32P-UTP for 10 min. RNA was harvested and hybridized to plasmids described in C dot-blotted onto Hybond-N+ membrane. For the NOY504 strain, cultures were incubated at 37°C for 6 h. (E) Quantitation of the results in D.

Figure 4.

Figure 4.

Quantitation of the number of rRNA transcripts per gene in chromatin spreads indicates that depletion of the t-Utps leads to a reduction in pre-rRNA transcription. Each strain carries the gene encoding the indicated protein under the control of a GAL promoter, allowing for conditional expression. The parent strain is YPH499. The boxes indicate t-Utps. (A_–_D) Examples of chromatin spreads from the indicated strains. The Mpp10, Utp4, and Utp10 proteins were depleted by growth in glucose for 6 h. (E) Results from quantitation of the number of rRNA transcripts per gene upon depletion of the indicated proteins. The average number of transcripts per gene for 32 genes for each strain is shown ± standard deviation.

Figure 5.

Figure 5.

The t-Utps are closely r-chromatin-associated. The indicated proteins were HA-tagged by integration of the tag at the genomic locus. YPH499 is the parental, untagged strain. Chromatin immunoprecipitations (ChIPs) with anti-HA antibodies (IP) (except for Rpa190, which was immunoprecipitated via the TAP tag with IgG beads) or with beads alone (B) were performed as described in Materials and Methods. R-chromatin association was assessed by assaying for the presence of immunoprecipitated 5′ETS sequences using PCR. The t-Utps are boxed. (A) ChIPs of tagged proteins required for ribosome biogenesis. Rpf2 and Imp4 are RNA-binding proteins containing the σ70-like motif, and are required for LSU and SSU biogenesis, respectively. Imp4, Rrp9, Utp3, and Utp7 are components of the SSU processome whose depletion does not affect transcription. Net1 is an RNA polymerase I-associated protein, and Rpa190 is a subunit of RNA polymerase I; they were used here as positive controls. Utp5, Utp8, Utp9, Utp10, Utp15, and Utp17 are t-Utps. (B) Quantitation of the results in A. (C) The t-Utps persist in their r-chromatin association in the absence of rRNA transcription. The indicated proteins were HA-tagged in the YJV100 strain by chromosomal integration. In the YJV100 strain, the endogenous rDNA has been replaced with 20–25 copies of the rDNA under the control of a galactose-inducible, glucose-repressible (GAL) Pol II promoter (Venema et al. 1995). rRNA transcription was interrupted (7 h, D) or not (0 h, U) by growth in glucose. ChIP was performed with immunoprecipitations with anti-HA antibodies (IP) or beads alone (B). Coimmunoprecipitating rDNA was analyzed by PCR with primers specific to portions of the 5′ETS.

Figure 6.

Figure 6.

(A) The t-Utps remain in a complex in the absence of the SSU processome. Proteins in the YKW100 strain were tagged with both the HA (Nop1, Utp5, Utp8, Utp9, and Utp15) and TAP (Utp17) tags, as indicated, by integration at the genomic locus. The t-Utps are boxed. The U3 snoRNA was either not depleted (0 h) or depleted (24 h). Anti-HA immunoprecipitation was performed on extracts from either depleted (D) or undepleted (U) cells, and proteins were analyzed by Western blot with anti-Mpp10 antibodies (to detect Mpp10) and with PAP (to detect the TAP tag on Utp17; top panel). (Bottom panel) Depletion of the SSU processome does not affect levels of SSU processome proteins. The U3 snoRNA was depleted (24 h, D) or not (0 h, U), and total protein levels were analyzed by Western blot with anti-Mpp10 antibodies and anti-HA antibodies (to detect the HA-tagged proteins). (B) Sedimentation profile of the t-Utps upon depletion of the SSU processome. Extracts from an HA-Utp8, TAP-Utp17-tagged YKW100 strain where the U3 snoRNA was either depleted (24 h) or not (0 h) were run on 10%–47% sucrose gradient. Fractions were analyzed by Western blot with anti-HA antibodies (to detect Utp8) or PAP (to detect Utp17). (C) The t-Utps remain in a complex in the absence of rRNA transcription. The indicated proteins were HA-tagged in the YJV100 strain by chromosomal integration. In the YJV100 strain, the endogenous rDNA was replaced with 20–25 copies of the rDNA under the control of a galactose-inducible, glucose-repressible (GAL) Pol II promoter. rRNA transcription was interrupted (7 h, D) or not (0 h, U) by growth in glucose. Anti-HA immunoprecipitations were performed on cell extracts, and coimmunoprecipitating proteins were analyzed by Western blot with anti-Mpp10 and anti-HA antibodies, and with PAP (to detect the TAP tag on Utp17). (D) Sedimentation profile of the t-Utps before and after interruption of transcription. Extracts from an HA-Utp8, TAP-Utp17-tagged YJV100 strain where shift to glucose (6 h) interrupted transcription were run on 10%–50% sucrose gradient. Fractions were analyzed by Western blot with anti-HA antibodies (to detect Utp8) or PAP (to detect Utp17).

Figure 7.

Figure 7.

(A) The t-Utps coimmunoprecipitate pre-18S rRNAs. The indicated proteins were HA-tagged by integration of the tag at the genomic locus. Boxes indicate t-Utps. “B” indicates immunoprecipitations performed with beads alone (no antibody), “IP” indicates immunoprecipitations performed with anti-HA antibody. Coimmunoprecipitating RNAs were isolated and analyzed by Northern blot with oligonucleotides complementary to the pre-rRNAs (oligos c, b, and e; Fig. 1). Pre-rRNA species were determined by their size relative to total RNA extracted by the hot phenol method from the parental YPH499 strain (data not shown). (B) The t-Utps coimmunoprecipitate pre-18S rRNAs in the absence of the U3 snoRNA. The indicated t-Utps were HA-tagged in the YKW100 strain. Anti-HA immunoprecipitations were performed on extracts from yeast depleted (D) or not (U) of the U3 snoRNA by growth in glucose. Coimmunoprecipitating RNAs were isolated and analyzed by Northern blot with an oligonucleotide complementary to pre-rRNAs (oligo c; Fig. 1).

Figure 8.

Figure 8.

Model linking rRNA transcription and pre-rRNA processing via components of the SSU processome. Association of the t-Utps with the r-chromatin is required for efficient transcription, and allows their assembly onto the nascent rRNA transcript. The U3 snoRNP then associates with the rRNA via base-pairing interactions, additional proteins join, and the SSU processome is formed. As a result, the pre-18S rRNA becomes accurately folded and ready for rRNA processing.

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