Gradual processing of the ITS1 from the nucleolus to the cytoplasm during synthesis of the human 18S rRNA

Abstract

Defects in ribosome biogenesis trigger stress response pathways, which perturb cell proliferation and differentiation in several genetic diseases. In Diamond–Blackfan anemia (DBA), a congenital erythroblastopenia, mutations in ribosomal protein genes often interfere with the processing of the internal transcribed spacer 1 (ITS1), the mechanism of which remains elusive in human cells. Using loss-of-function experiments and extensive RNA analysis, we have defined the precise position of the endonucleolytic cleavage E in the ITS1, which generates the 18S-E intermediate, the last precursor to the 18S rRNA. Unexpectedly, this cleavage is followed by 3′–5′ exonucleolytic trimming of the 18S-E precursor during nuclear export of the pre-40S particle, which sets a new mechanism for 18S rRNA formation clearly different from that established in yeast. In addition, cleavage at site E is also followed by 5′–3′ exonucleolytic trimming of the ITS1 by exonuclease XRN2. Perturbation of this step on knockdown of the large subunit ribosomal protein RPL26, which was recently associated to DBA, reveals the putative role of a highly conserved cis-acting sequence in ITS1 processing. These data cast new light on the original mechanism of ITS1 elimination in human cells and provide a mechanistic framework to further study the interplay of DBA-linked ribosomal proteins in this process.

Human NF-κB repressing factor acts as a stress-regulated switch for ribosomal RNA processing and nucleolar homeostasis surveillance

“…This process was mainly characterized in yeast and, surprisingly, only recently investigated in human cells, revealing that pre-rRNA processing pathways are notably different in metazoan: ∼27% of human factors have distinct or additional functions in pre-rRNA processing compared with their yeast orthologs, and several pre-RNA processing factors have no yeast homolog (26,29). XRN2 (homolog of yeast XRN2/Rat1) plays a major role in rRNA maturation, coordinating the optimal order of multiple pre-rRNA cleavages (25); it is essential for degradation of 5′-extended 45.5S and 34.5S pre-rRNAs forms, it removes ITS-1-derived extensions to generate 32S from 32.5S pre-rRNA, and it promotes decay of 5′-01, 5′-A0, and E2 fragments, generated by endonucleolytic cleavages in the 5′ETS/ITS1 region in human cells (27)(28)(29) (Fig. 4A).…”

hUTP24 is essential for processing of the human rRNA precursor at site A1, but not at site A0

“…7,8 Thus, there are additional processing sites within human 47S pre-rRNA. [9][10][11] Moreover, in some cases the functions of homologous proteins involved in ribosome synthesis in both species are not equivalent. [12][13][14] Furthermore, alternative pre-rRNA processing pathways are utilized in parallel in both model organisms.…”

“…Finally, there are processing events in humans, but not yeast, that occur either through endonucleolytic cleavage or by exoribonucleolytic maturation. 11,16 Processing of human 47S pre-rRNA begins with primary cleavage at site A 0 (also known as 01) in the 5 0 -ETS, which encompasses endoribonucleolytic cleavage sites at 2 adjacent regions between nucleotides 414 and 422 (Fig. 1A).…”

Qu’en est-il de la biogenèse des ribosomes chez l’homme ?

“…Ce dernier clivage a lieu dans le cytoplasme. De la même manière que pour le 21S, le pré-ARNr 18S-E subit un rognage 3'-5' préalablement au clivage au site 3 [18]. D'autre part, le pré-ARNr 32S est clivé au site 3' dans l'ITS2, générant l'ARNr mature 28S et le pré-ARNr 12S.…”

“…Ces derniers suivent alors les étapes de clivage décrites précédemment. Dans certaines conditions, le clivage au niveau de l'ITS1 se produit au site E plutôt qu'au site 2 [17,18], générant alors le pré-ARNr 18S-E et un précurseur appelé 36S ( Figure 2C Certains facteurs d'assemblage servent d'adaptateurs pour recruter les exportines et ont ainsi un rôle dans le contrôle de qualité. Dans le cas des précurseurs de la grande sous-unité, c'est la protéine NMD3 qui recrute l'exportine CRM1 (chromosome region maintenance 1; also referred to as exportin1 or Xpo1) [24], et, pour les pré-40S, ce sont les facteurs d'assemblage RIOK2 (RIO kinase 2) et TSR1 qui seraient requis pour lier CRM1 [16,25].…”