The Rea1 Tadpole Loses Its Tail

More than 170 assembly factors aid the construction and maturation of yeast ribosomes. After these factors' functions are completed, they must be released from preribosomes. In this issue, Ulbrich et al. (2009) describe a mechanochemical process through which the AAA ATPase Rea1 induces release of an assembly protein complex from preribosomes

Rapidly evolving protointrons in Saccharomyces genomes revealed by a hungry spliceosome.

Introns are a prevalent feature of eukaryotic genomes, yet their origins and contributions to genome function and evolution remain mysterious. In budding yeast, repression of the highly transcribed intron-containing ribosomal protein genes (RPGs) globally increases splicing of non-RPG transcripts through reduced competition for the spliceosome. We show that under these "hungry spliceosome" conditions, splicing occurs at more than 150 previously unannotated locations we call protointrons that do not overlap known introns. Protointrons use a less constrained set of splice sites and branchpoints than standard introns, including in one case AT-AC in place of GT-AG. Protointrons are not conserved in all closely related species, suggesting that most are not under positive selection and are fated to disappear. Some are found in non-coding RNAs (e. g. CUTs and SUTs), where they may contribute to the creation of new genes. Others are found across boundaries between noncoding and coding sequences, or within coding sequences, where they offer pathways to the creation of new protein variants, or new regulatory controls for existing genes. We define protointrons as (1) nonconserved intron-like sequences that are (2) infrequently spliced, and importantly (3) are not currently understood to contribute to gene expression or regulation in the way that standard introns function. A very few protointrons in S. cerevisiae challenge this classification by their increased splicing frequency and potential function, consistent with the proposed evolutionary process of "intronization", whereby new standard introns are created. This snapshot of intron evolution highlights the important role of the spliceosome in the expansion of transcribed genomic sequence space, providing a pathway for the rare events that may lead to the birth of new eukaryotic genes and the refinement of existing gene function

Yeast Polypeptide Exit Tunnel Ribosomal Proteins L17, L35, and L37 are Necessary to Recruit Late-assembling Factors Required for 27SB Pre-rRNA Processing

Ribosome synthesis involves the coordinated folding and processing of pre-rRNAs with assembly of ribosomal proteins. In eukaryotes, these events are facilitated by trans-acting factors that propel ribosome maturation from the nucleolus to the cytoplasm. However, there is a gap in understanding how ribosomal proteins configure pre-ribosomes in vivo to enable processing to occur. Here, we have examined the role of adjacent yeast r-proteins L17, L35 and L37 in folding and processing of pre-rRNAs, and binding of other proteins within assembling ribosomes. These three essential ribosomal proteins, which surround the polypeptide exit tunnel, are required for 60S subunit formation as a consequence of their role in removal of the ITS2 spacer from 27SB pre-rRNA. L17-, L35- and L37-depleted cells exhibit turnover of aberrant pre-60S assembly intermediates. Although the structure of ITS2 does not appear to be grossly affected in their absence, these three ribosomal proteins are necessary for efficient recruitment of factors required for 27SB pre-rRNA processing, namely, Nsa2 and Nog2, which associate with pre-60S ribosomal particles containing 27SB pre-rRNAs. Altogether, these data support that L17, L35 and L37 are specifically required for a recruiting step immediately preceding removal of ITS2

Ebp2 and Brx1 function cooperatively in 60S ribosomal subunit assembly in Saccharomyces cerevisiae

The yeast protein Ebp2 is required for early steps in production of 60S ribosomal subunits. To search for cofactors with which Ebp2 functions, or substrates on which it acts, we screened for mutants that were synthetically lethal (sl) with the ebp2-14 mutation. Four different mutant alleles of the 60S ribosomal subunit assembly factor Brx1 were found. To investigate defects of the double mutant, we constructed strains conditional for the ebp2-14 brx1- synthetic lethal phenotype. These ebp2-14 brx1 mutants were defective in processing of 27S pre-rRNA and production of 60S subunits, under conditions where each single mutant was not. Ebp2 and Brx1 exhibit a strong two-hybrid interaction, which is eliminated by some combinations of brx1 and ebp2 mutations. In one such mutant, Ebp2 and Brx1 can still associate with pre-ribosomes, but subunit maturation is perturbed. Depletion of either Ebp2 or Brx1 revealed that Brx1 requires Ebp2 for its stable association with pre-ribosomes, but Ebp2 does not depend on the presence of Brx1 to enter pre-ribosomes. These results suggest that assembly of 60S ribosomal subunits requires cooperation of Ebp2 with Brx1, together with other molecules present in pre-ribosomes, potentially including several found in assembly subcomplexes with Brx1 and Ebp2

Hierarchical Association of Subcomplexes Drives pre-rRNA Folding during Ribosome Biogenesis in <i>Saccharomyces cerevisiae</i>

<p>Construction of eukaryotic ribosomes requires at least 180 assembly factors. These proteins are thought to drive forward the progressive folding and processing of pre-rRNAs, and binding of ribosomal proteins. Initial characterizations have revealed in which steps of pre-rRNA processing most of these factors function, but only recently have investigations begun to understand how each of these proteins contributes to folding of the nascent rRNA.</p> <p>Work from our lab, part of which is presented in this thesis, has established an association hierarchy between 12 assembly factors and ribosomal proteins necessary for processing 27SA₃ pre-rRNA. The most up-stream factors in this pathway are present in a subcomplex of four assembly factors and two ribosomal proteins. I focused on the understanding the roles of two of these proteins, Nop12 and Pwp1, in ribosome biogenesis. I investigated their timing of association with pre-ribosomes, their role in recruiting other proteins, and the effects on pre-rRNA folding in their absence. By doing so, I show that the effects observed on pre-rRNA processing in the absence of Nop12 and Pwp1 are not simply due to pre-ribosomes lacking a complete inventory of proteins, but rather due to a failure to properly fold the rRNA.</p> <p>I also investigated the function of the DEAD-box protein Drs1 in ribosome biogenesis and show that it functions in two consecutive steps of ribosome assembly. Furthermore, physical and genetic interactions reveal that Drs1 is recruited to pre- ribosomes by a subcomplex of assembly factors that function in the same step of biogenesis.</p> <p>Most importantly, in the course of investigating Drs1, I found that disruption of ribosome biogenesis results in a shift from pre-rRNAs being processed co-transcriptionally, to being processed post-transcriptionally. When this occurs, there is a breakdown of the global hierarchy of ribosome biogenesis. I show that normally late associating proteins associate with pre-ribosomes early in the biogenesis pathway, prior to any pre-rRNA processing steps occurring. Furthermore, I show this shift from co- to post-transcriptional processing is related to cell growth rates, and pre-rRNAs processed post-transcriptionally proceed down an alternative maturation pathway. These results have drastic implications for how we view the overall hierarchy of ribosome biogenesis.</p

Ribosome assembly factors Pwp1 and Nop12 are important for folding of 5.8S rRNA during ribosome biogenesis in Saccharomyces cerevisiae.

Previous work from our lab suggests that a group of interdependent assembly factors (A(3) factors) is necessary to create early, stable preribosomes. Many of these proteins bind at or near internal transcribed spacer 2 (ITS2), but in their absence, ITS1 is not removed from rRNA, suggesting long-range communication between these two spacers. By comparing the nonessential assembly factors Nop12 and Pwp1, we show that misfolding of rRNA is sufficient to perturb early steps of biogenesis, but it is the lack of A(3) factors that results in turnover of early preribosomes. Deletion of NOP12 significantly inhibits 27SA(3) pre-rRNA processing, even though the A(3) factors are present in preribosomes. Furthermore, pre-rRNAs are stable, indicating that the block in processing is not sufficient to trigger turnover. This is in contrast to the absence of Pwp1, in which the A(3) factors are not present and pre-rRNAs are unstable. In vivo RNA structure probing revealed that the pre-rRNA processing defects are due to misfolding of 5.8S rRNA. In the absence of Nop12 and Pwp1, rRNA helix 5 is not stably formed. Interestingly, the absence of Nop12 results in the formation of an alternative yet unproductive helix 5 when cells are grown at low temperatures.</p