Jorge Perez-Fernandez

The 90S preribosome is a multimodular structure that is assembled through a hierarchical mechanism.

ABSTRACT The 90S preribosomal particle is required for the production of the 18S rRNA from a pre-rRNA precursor. Despite the identification of the protein components of this particle, its mechanism of assembly and structural design remain unknown. In this work, we have combined biochemical studies, proteomic techniques, and bioinformatic analyses to shed light into the rules of assembly of the yeast 90S preribosome. Our results indicate that several protein subcomplexes work as discrete assembly subunits that bind in defined steps to the 35S pre-rRNA. The assembly of the t-UTP subunit is an essential step for the engagement of at least five additional subunits in two separate, and mutually independent, assembling routes. One of these routes leads to the formation of an assembly intermediate composed of the U3 snoRNP, the Pwp2p/UTP-B, subunit and the Mpp10p complex. The other assembly route involves the stepwise binding of Rrp5p and the UTP-C subunit. We also report the use of a bioinformatic approach that provides a model for the topological arrangement of protein components within the fully assembled particle. Together, our data identify the mechanism of assembly of the 90S preribosome and offer novel information about its internal architecture.

Chromatin states at ribosomal DNA loci

Eukaryotic transcription of ribosomal RNAs (rRNAs) by RNA polymerase I can account for more than half of the total cellular transcripts depending on organism and growth condition. To support this level of expression, eukaryotic rRNA genes are present in multiple copies. Interestingly, these genes co-exist in different chromatin states that may differ significantly in their nucleosome content and generally correlate well with transcriptional activity. Here we review how these chromatin states have been discovered and characterized focusing particularly on their structural protein components. The establishment and maintenance of rRNA gene chromatin states and their impact on rRNA synthesis are discussed. This article is part of a Special Issue entitled: Transcription by Odd Pols.

Elucidation of the assembly events required for the recruitment of Utp20, Imp4 and Bms1 onto nascent pre-ribosomes

The 90S pre-ribosome, also known as the small subunit (SSU) processome, is a large multisubunit particle required for the production of the 18S rRNA from a pre-rRNA precursor. Recently, it has been shown that the formation of this particle entails the initial association of the tUTP subunit with the nascent pre-RNA and, subsequently, the binding of Rrp5/UTP-C and U3 snoRNP/UTP-B subunits in two independent assembly branches. However, the mode of assembly of other 90S pre-ribosome components remains obscure as yet. In this study, we have investigated the assembly of three proteins (Utp20, Imp4 and Bms1) previously regarded as potential nucleating factors of the 90S particle. Here, we demonstrate that the loading of those three proteins onto the pre-rRNA takes place independently of Rrp5/UTP-C and, instead, occurs downstream of the tUTP and U3/UTP-B subcomplexes. We also demonstrate that Bms1 and Utp20 are required for the recruitment of a subset of proteins to nascent pre-ribosomes. Finally, we show that proteins associated through secondary steps condition the stability of the two assembly branches in partially assembled pre-ribosomes. These results provide new information about the functional relationships among 90S particle components and the events that are required for their stepwise incorporation onto the primary pre-rRNA.

Studies on the Assembly Characteristics of Large Subunit Ribosomal Proteins in S. cerevisae

During the assembly process of ribosomal subunits, their structural components, the ribosomal RNAs (rRNAs) and the ribosomal proteins (r-proteins) have to join together in a highly dynamic and defined manner to enable the efficient formation of functional ribosomes. In this work, the assembly of large ribosomal subunit (LSU) r-proteins from the eukaryote S. cerevisiae was systematically investigated. Groups of LSU r-proteins with specific assembly characteristics were detected by comparing the protein composition of affinity purified early, middle, late or mature LSU (precursor) particles by semi-quantitative mass spectrometry. The impact of yeast LSU r-proteins rpL25, rpL2, rpL43, and rpL21 on the composition of intermediate to late nuclear LSU precursors was analyzed in more detail. Effects of these proteins on the assembly states of other r-proteins and on the transient LSU precursor association of several ribosome biogenesis factors, including Nog2, Rsa4 and Nop53, are discussed.

The Reb1-homologue Ydr026c/Nsi1 is required for efficient RNA polymerase I termination in yeast.

Several DNA cis‐elements and trans‐acting factors were described to be involved in transcription termination and to release the elongating RNA polymerases from their templates. Different models for the molecular mechanism of transcription termination have been suggested for eukaryotic RNA polymerase I (Pol I) from results of in vitro and in vivo experiments. To analyse the molecular requirements for yeast RNA Pol I termination, an in vivo approach was used in which efficient termination resulted in growth inhibition. This led to the identification of a Myb‐like protein, Ydr026c, as bona fide termination factor, now designated Nsi1 (NTS1 silencing protein 1), since it was very recently described as silencing factor of ribosomal DNA. Possible Nsi1 functions in regard to the mechanism of transcription termination are discussed.

Interrelationships between Yeast Ribosomal Protein Assembly Events and Transient Ribosome Biogenesis Factors Interactions in Early Pre-Ribosomes

Early steps of eukaryotic ribosome biogenesis require a large set of ribosome biogenesis factors which transiently interact with nascent rRNA precursors (pre-rRNA). Most likely, concomitant with that initial contacts between ribosomal proteins (r-proteins) and ribosome precursors (pre-ribosomes) are established which are converted into robust interactions between pre-rRNA and r-proteins during the course of ribosome maturation. Here we analysed the interrelationship between r-protein assembly events and the transient interactions of ribosome biogenesis factors with early pre-ribosomal intermediates termed 90S pre-ribosomes or small ribosomal subunit (SSU) processome in yeast cells. We observed that components of the SSU processome UTP-A and UTP-B sub-modules were recruited to early pre-ribosomes independently of all tested r-proteins. On the other hand, groups of SSU processome components were identified whose association with early pre-ribosomes was affected by specific r-protein assembly events in the head-platform interface of the SSU. One of these components, Noc4p, appeared to be itself required for robust incorporation of r-proteins into the SSU head domain. Altogether, the data reveal an emerging network of specific interrelationships between local r-protein assembly events and the functional interactions of SSU processome components with early pre-ribosomes. They point towards some of these components being transient primary pre-rRNA in vivo binders and towards a role for others in coordinating the assembly of major SSU domains.

RNA polymerase I termination: Where is the end?

The synthesis of ribosomal RNA (rRNA) precursor molecules by RNA polymerase I (Pol I) terminates with the dissociation of the protein-DNA-RNA ternary complex. Based on in vitro results the mechanism of Pol I termination appeared initially to be rather conserved and simple until this process was more thoroughly re-investigated in vivo. A picture emerged that Pol I termination seems to be connected to co-transcriptional processing, re-initiation of transcription and, possibly, other processes downstream of Pol I transcription units. In this article, our current understanding of the mechanism of Pol I termination and how this process might be implicated in other biological processes in yeast and mammals is summarized and discussed. This article is part of a Special Issue entitled: Transcription by Odd Pols.

Regulation of ribosomal RNA production by RNA polymerase I: does elongation come first?

Ribosomal RNA (rRNA) production represents the most active transcription in the cell. Synthesis of the large rRNA precursors (35–47S) can be achieved by up to 150 RNA polymerase I (Pol I) enzymes simultaneously transcribing each rRNA gene. In this paper, we present recent advances made in understanding the regulatory mechanisms that control elongation. Built-in Pol I elongation factors, such as Rpa34/Rpa49 in budding yeast and PAF53/CAST in humans, are instrumental to the extremely high rate of rRNA production per gene. rRNA elongation mechanisms are intrinsically linked to chromatin structure and to the higher-order organization of the rRNA genes (rDNA). Factors such as Hmo1 in yeast and UBF1 in humans are key players in rDNA chromatin structure in vivo. Finally, elongation factors known to regulate messengers RNA production by RNA polymerase II are also involved in rRNA production and work cooperatively with Rpa49 in vivo.

In Vitro Reconstitution of Yeast tUTP/UTP A and UTP B Subcomplexes Provides New Insights into Their Modular Architecture

Eukaryotic ribosome biogenesis is a multistep process involving more than 150 biogenesis factors, which interact transiently with pre-ribosomal particles to promote their maturation. Some of these auxiliary proteins have been isolated in complexes found separate from the ribosomal environment. Among them, are 3 large UTP subcomplexes containing 6 or 7 protein subunits which are involved in the early steps of ribosome biogenesis. The composition of the UTP subcomplexes and the network of binary interactions between protein subunits have been analyzed previously. To obtain further insights into the structural and biochemical properties of UTP subcomplexes, we established a heterologous expression system to allow reconstitution of the yeast tUTP/UTP A and UTP B subcomplexes from their candidate subunits. The results of a series of reconstitution experiments involving different combinations of protein subunits are in good agreement with most of the previously observed binary interactions. Moreover, in combination with additional biochemical analyses, several stable building blocks of the UTP subcomplexes were identified. Based on these findings, we present a refined model of the tUTP/UTP A and UTP B architecture.

Purification of specific chromatin domains from single-copy gene loci in Saccharomyces cerevisiae.

Most methods currently available for the analysis of chromatin in vivo rely on a priori knowledge of putative chromatin components or their posttranslational modification state. The isolation of defined native chromosomal regions provides an attractive alternative to obtain a largely unbiased molecular description of chromatin. Here, we describe a strategy combining site-specific recombination at the chromosome with an efficient tandem affinity purification protocol to isolate a single-copy gene locus from the yeast Saccharomyces cerevisiae. The method allows robust enrichment of a targeted chromatin domain, making it amenable to compositional, structural, and biochemical analyses. This technique appears to be suitable to obtain a detailed description of chromatin composition and specific posttranslational histone modification state at virtually any genomic locus in yeast.