Deletion of Rnt1p Alters the Proportion of Open versus Closed rRNA Gene Repeats in Yeast

Abstract

In Saccharomyces cerevisiae, the double-stranded-RNA-specific RNase III (Rnt1p) is required for the processing of pre-rRNA and coprecipitates with transcriptionally active rRNA gene repeats. Here we show that Rnt1p physically interacts with RNA polymerase I (RNAPI) and its deletion decreases the transcription of the rRNA gene and increases the number of rRNA genes with an open chromatin structure. In contrast, depletion of ribosomal proteins or factors that impair RNAPI termination did not increase the number of open rRNA gene repeats, suggesting that changes in the ratio of open and closed rRNA gene chromatin is not due to a nonspecific response to ribosome depletion or impaired termination. The results demonstrate that defects in pre-rRNA processing can influence the chromatin structure of the rRNA gene arrays and reveal links among the rRNA gene chromatin, transcription, and processing.In Saccharomyces cerevisiae, ribosomes are formed of 78 different ribosomal proteins assembled around four rRNAs. The synthesis of these essential protein factories involves three RNA polymerases and several processing factors (70). The ribosomal protein genes are transcribed by RNA polymerase II, while the rRNAs are transcribed by RNA polymerase I (RNAPI), with the exception of the 5S rRNA that is produced by RNA polymerase III. RNAPI produces the 18S, 5.8S, and 25S rRNAs as a single transcript containing two external transcribed spacers (ETS1 and -2) and two internal transcribed spacers (ITS1 and -2). Once transcribed, the rRNA precursor is processed to form the ribosome small subunit containing the 18S rRNA and the ribosome large subunit (LSU) containing the 5.8S and 25S rRNAs (69). RNAPI ( Fig. 1) is composed of five subunits common to all RNA polymerases, two subunits shared with RNA polymerase III, and seven unique subunits (53). All of the shared subunits are essential for yeast growth, as are the two largest unique subunits. The five remaining unique subunits are dispensable; however, the loss of Rpa12p causes temperature sensitivity and impairs RNAPI termination (21, 52). The last four RNAPIspecific subunits form two distinct complexes; one contains the Rpa34p and Rpa49p subunits, and the other contains the Rpa14p and Rpa43p subunits. The Rpa34p-Rpa49p complex is located near Rpa12p (Fig.

Efficient termination of transcription by RNA polymerase I requires the 5′ exonuclease Rat1 in yeast

“…Rnt1 activity influences rDNA transcription and chromatin structure (Catala et al 2008), consistent with cotranscriptional activity. A rnt1⌬ strain showed increased read-through of T1 with most Pol I stopping at T2 (Reeder et al 1999;Prescott et al 2004), and readthrough transcripts extending to T2 were also detected Fig.…”

“…in rnt1-ts strains (Catala et al 2008), indicating that cotranscriptional cleavage is important for efficient termination at T1. Rnt1 cleavage generates products with 5Ј-monophosphate termini, the preferred substrates for Rat1 (Stevens and Poole 1995), which was reported previously to participate in the degradation of the 3Ј-cleavage product (Kufel et al 1999), raising the interesting possibility that Rat1 might also "torpedo" Pol I during transcription termination.…”

“…2A; Kufel et al 1999). Rnt1 was detected at the site of rRNA transcription (Henras et al 2004), and can interact physically with Pol I subunits including Rpa12 (Catala et al 2008). Moreover, loss of Pol I promoter.…”

Transcription termination by nuclear RNA polymerases

“…Deletion of Rnt1 or mutation disrupting its interaction with RNAPI altered the chromatin conformation of rDNA by increasing the number of open rRNA gene repeats. However, deletion of RPA12 alone had no effect on chromatin opening and pre-rRNA processing (Catala et al 2008). Interestingly, as shown for RNAPII termination (Alen et al 2002), chromatin remodeling factors are also involved in RNAPI termination.…”

“…Inactivation of Rnt1 causes read-through at T1, indicating that, as with protein-coding genes, cotranscriptional cleavage is important for efficient termination Prescott et al 2004;Kawauchi et al 2008). Recently, Catala et al (2008) showed that Rnt1 interacts with two RNAPIspecific subunits, Rpa12 and Rpa34. Rpa12 shares sequence similarity with the small RNAPII subunit Rpb9 and with C11 of RNAPIII.…”