The nucleolus and rRNA genes


Responsible : Julio SAEZ VASQUEZ (DR2 CNRS)

Team :

BRANDO-GOT Sophie - Adjoint technique UPVD

COMELLA Pascale - Maître de conférences UPVD

DARRIERE Tommy - CDD chercheur CNRS

DE BURES Anne - Technicienne UPVD

SAEZ-VASQUEZ Julio - Chercheur CNRS

 

Context

The nucleolus is the most prominent nuclear structure. For a long time, it was essentially considered as a ribosome biogenesis factory, constitutively generating the basic protein synthesis machinery. It is now clear that this subnuclear compartment plays wider roles, notably in cellular responses to intrinsic and environmental changes. The nucleolus also has novel and poorly characterized functions. In particular, in protein sequestering via interaction with other proteins and/or long non-coding RNAs. Nucleolar sequestration represents a new posttranslational regulatory mechanism that could negatively or positively regulate the activity of targeted proteins (see figure).

The driving force for nucleolar assembly is gene transcription of 45S rRNA genes (45S rDNA) and processing of 45S rRNA precursors (pre-rRNA), transcribed by RNA polymerase I (Pol I), and the assembly of ribosome particles. Eukaryotic cells contain hundreds or thousands of 45S rDNA copies localized in Nucleolus Organizer Regions (NORs). However, only a fraction of these genes is transcriptionally active or competent for RNA Pol I transcription. Permissive or transcriptionally-active rRNA genes (euchromatin) are located in the nucleolus whereas repressive or transcriptionally-inactive rRNA genes (heterochromatin) remain in the nucleoplasm, and are merely associated with the nucleolus. Thus, part of the NOR is structured as a knob of condensed chromatin, whereas the rest forms extended loops of rDNA genes from which the nucleolus originates. In Arabidopsis thaliana (Col-0), 45S rDNA euchromatin loops from NOR4 are extensively transcribed by RNA pol I, whereas highly condensed 45S rDNA, heterochromatin, from NOR2 is usually transcriptionally silent. Thus the expression of rDNAs/NORs involves epigenetic regulatory mechanisms.

In the nucleolus, transcription of 45S rRNA genes is coupled to processing of pre-rRNA. Maturation of the pre-rRNA to obtain the mature 18S, 5.8S and 25S rRNAs involves the participation of the different endo- and exonucleases, for the elimination of spacer sequences, and the covalent modification of specific nucleotides in the rRNA. The mature 18S RNAs and the 5.8S and 25S rRNAs respectively associate with the 40S and 60S subunits of the ribosomes. The most abundant modifications of rRNA are methylation of ribose (2'-O-methylation) and isomerization of uridine (pseudouridylation), directed respectively by C/D-box and H/ACA-box small nucleolar (snoRNA) RNAs. The impact of rRNA modifications on the structure and/or function of ribosomes is not yet known in plants.

Current work

On the one hand, the team aims to characterize molecular and functional connections between nucleolar activity and temperature stress responses in Arabidopsis thaliana. First, we seek to identify and understand nucleolar sequestration mechanisms of proteins through interactions with non-coding RNAs, transcribed from intergenic regions (IGS) of ribosomal RNA genes (lGS-ncRNAs). These interactions form the basis of a new post-translational regulatory mechanism that regulates nuclear and / or nucleolar protein activity by retention/sequestration in the nucleolus. Second, we seek to study the impact of specific pathways of pre-rRNA processing and (2'-O-methylation) modifications of rRNA on ribosome translation activity. In other words, we study how rRNA dynamics could contribute to the production of (structurally and functionally) heterogeneous ribosomes in plants.

On the other hand, with the objective of identifying the endonuclease involved in the processing of ribosomal RNAs; a few years ago, we initiated the characterization of a family of RNaseIII type proteins in Arabidopsis. The Arabidopsis genome codes for 7 protein sequences containing an RNaseIII motif. Four code for Dicer type proteins: DCL1, -2, -3 and -4 while the other three belong to another small family: RTL1, RTL2 and RTL3. We have demonstrated that RTLs are involved in the maturation of rRNAs and / or RNA precursors of miRNAs and siRNA, recognized and cleaved by DCL1 and DCL2-4 respectively. In this context, we try to identify RNA and protein molecular bases that regulate and determine cleavage activity of RTLs. For this, we develop biochemical, genetic, structural and functional approaches.

The research work of the team is mostly carried out within the framework of national projects (ANR EpiRNAse, SUBCELIF and RiboStress), international projects with Spain (PAI Picasso), Japan (JSPS / CNRS) and Czech Republic (Campus France Barrande) and in collaboration with numerous laboratories in France and abroad (Europe, Asia and the United States).

 



Publications :

31, 1945–1967 (2019).

 

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