Reference : UMR5077-BENALB-003
Workplace : TOULOUSE
Date of publication : Tuesday, June 21, 2022
Scientific Responsible name : benjamin Albert
Type of Contract : PhD Student contract / Thesis offer
Contract Period : 36 months
Start date of the thesis : 1 October 2022
Proportion of work : Full time
Remuneration : 2 135,00 € gross monthly
Description of the thesis topic
Eukaryotic ribosome consists of two subunits formed by 79 ribosomal proteins (RPs) and 4 ribosomal RNAs (rRNAs)(1). Ribosome production starts with the synthesis of a large ribosomal RNA precursor (pre-rRNA) by RNA polymerase I (RNAPI). Around 80 small nucleolar ribonucleoprotein particles (snoRNPs) and 150 assembly and maturation factors (AMFs) are required to convert this precursor into a mature ribosome. This process is initiated during transcription: the nascent pre-rRNA associates co-transcriptionally with snoRNPs and some RPs and AMFs to form precursor particles called pre-ribosomes. Very early, numerous nucleotides of the pre-rRNAs are chemically modified by two families of snoRNPs: box C/D and box H/ACA snoRNPs, known to catalyse ribose methylations and isomerization of uridines into pseudouridines, respectively. Their snoRNA components base‐pair with the unfolded pre‐rRNA at the vicinity of the nucleotides to be modified, thereby guiding the enzymatic modifications catalyzed by one of the core proteins of the snoRNPs: the methyltransferase Nop1/Fibrillarin in box C/D snoRNPs and the pseudouridine synthase Cbf5/Dyskerin in H/ACA snoRNPs. The folding of rRNAs is directly impacted by nucleotide modifications since ribose methylations and pseudouridylations rigidify the RNA backbone (2). A few snoRNAs of both families (U3, U14, snR30 and snR10) do not function as modification guides but as chaperones in the folding of the pre-rRNA. Therefore, snoRNPs are required for both nucleotide modifications and proper folding of the pre-rRNA, two essential aspects of the maturation of this precursor into functional 18S, 5.8S, and 25S rRNAs.
The chronology of assembly of snoRNPs and AMFs with the nascent transcript is crucial to achieve proper rRNA folding and processing. Given their importance in the very early stages, most snoRNPs should interact with the unfolded pre-rRNA prior to recruitment of the AMFs and RPs in these regions, and prior to rRNA folding. This process is very efficient since more than 90% of the nucleotides targeted by either C/D or H/ACA snoRNPs are modified in the mature rRNAs, as shown using RiboMethSeq and HydraPsiSeq approaches, respectively (3,4). How the very early recruitment of various snoRNPs is achieved has remained an enigma.
We propose here to tackle a very important and so far unanswered question concerning the earliest stages of ribosome biogenesis in eukaryotes: when and how are snoRNPs recruited onto the nascent pre-rRNA. We anticipate that efficient snoRNPs recruitment and dissociation depends not only on base-pairing between snoRNAs and rRNAs, but also on a complex interaction network between the snoRNPs, RNAPI and the nascent transcript.
The main challenge in this PHD will be to decipher the mechanisms allowing robust snoRNP recruitment to rRNA nascent transcript.
Ribosome production in eukaryotes is initiated by the transcription by RNA polymerase I of a pre-ribosomal RNA (pre-rRNA), precursor to the 18S, 5.8S and 25S/28S ribosomal RNAs (rRNAs). This pre-rRNA associates co-transcriptionally with ribosomal proteins, small nucleolar ribonucleoprotein particles (snoRNPs) and non-ribosomal proteins to yield a nascent pre-ribosomal particle from which the early pre-40S particle containing the 18S rRNA sequence is split mostly co-transcriptionally. The early pre-60S particle assembled onto the 5.8S and 25S sequences is released subsequently following transcription termination. These particles then follow independent maturation pathways, first in the nucleus and finally in the cytoplasm. Within this series of pre-ribosomal particles, the pre-rRNAs are chemically modified and progressively processed by endo- and exoribonucleases to release the mature rRNAs. Hundreds of snoRNPs and more than 200 non-ribosomal proteins are involved in ribosome synthesis in eukaryotes. The molecular functions of most of these co-factors, several of which are enzymes, remain elusive. Our aims are to understand the precise molecular functions and modes of regulation of several key non-ribosomal factors involved in the co- and/or post-transcriptional steps of ribosome synthesis, in particular energy-consuming enzymes.
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