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The 3’-5’ exoribonuclease ISG20L2 contributes to 3’ terminus maturation of 18S and 28S ribosomal RNAs

ABSTRACT: Ribosome biogenesis is a nodal process in cell growth and proliferation, and its dysfunction is associated to human congenital diseases and tumorigenesis. Despite extensive mechanistic characterization, several processing steps of human pre-ribosomal RNAs remain elusive. The 47S primary transcript contains 3 out of 4 ribosomal RNAs flanked by external (5’ETS, 3’ETS) and internal (ITS1, ITS2) transcribed spacers. The molecular processes leading to the removal of the 3’ETS, one of the earliest maturation steps, is not fully understood. Combining loss-of-function experiments and 3’-RACE high-throughput sequencing, we showed that the vertebrate-specific 3’-5’ exoribonuclease ISG20L2, a DEDDh RNase T superfamily member, is critical for efficient removal of the 3’ETS and formation of large ribosomal subunits. ISG20L2 inactivation led to accumulation of various forms of 3’-extented pre-rRNAs and disorganized PDFC, a sub-nucleolar compartment recently linked to 3’ETS processing. The function of ISG20L2 also extends to the trimming of ITS1 after endonucleolytic cleavage at site 2 and its knockdown reveals the precise location of site 2. Altogether, the present work uncovers the landscape of two pre-rRNA processing steps at nucleotide resolution, and restricts the function of ISG20L2 to the nucleoli where it contributes to the maturation of 18S and 28S 3’ ends.

ORGANISM(S): Homo sapiens

PROVIDER: GSE272872 | GEO | 2025/10/16

REPOSITORIES: GEO

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The 3’-5’ exoribonuclease ISG20L2 contributes to 3’ terminus maturation of 18S and 28S ribosomal RNAs (revision)

Project description:Ribosome biogenesis is a nodal process in cell growth and proliferation, and its dysfunction is associated to human congenital diseases and tumorigenesis. Despite extensive mechanistic characterization, several processing steps of human pre-ribosomal RNAs remain elusive. The 47S primary transcript contains 3 out of 4 ribosomal RNAs flanked by external (5’ETS, 3’ETS) and internal (ITS1, ITS2) transcribed spacers. The molecular processes leading to the removal of the 3’ETS, one of the earliest maturation steps, is not fully understood. Combining loss-of-function experiments and 3’-RACE high-throughput sequencing, we showed that the vertebrate-specific 3’-5’ exoribonuclease ISG20L2, a DEDDh RNase T superfamily member, is critical for efficient removal of the 3’ETS and formation of large ribosomal subunits. ISG20L2 inactivation led to accumulation of various forms of 3’-extented pre-rRNAs and disorganized PDFC, a sub-nucleolar compartment recently linked to 3’ETS processing. The function of ISG20L2 also extends to the trimming of ITS1 after endonucleolytic cleavage at site 2 and its knockdown reveals the precise location of site 2. Altogether, the present work uncovers the landscape of two pre-rRNA processing steps at nucleotide resolution, and restricts the function of ISG20L2 to the nucleoli where it contributes to the maturation of 18S and 28S 3’ ends.

2025-10-16 | GSE292922 | GEO

Effect of Nop53 depletion on the compositional changes during pre-60S maturation

Project description:The eukaryotic ribosome biogenesis is a highly orchestrated multistep process that starts at the nucleolus with the transcription of pre-rRNAs 5S and 35S. The latter comprises the mature 18S, 5.8S and 25S rRNAs separated by internal transcribed spacers (ITS1 and ITS2) and externally flanked by the 5’ETS and 3’ETS. The 35S pre-rRNA undergoes several co- and post-transcriptional processing events, which will enable the pre-60S and pre-40S particles to take independent maturation routes. Hundreds of assembly factors (AF) are required, being recruited and released hierarchically, for the proper folding of the rRNAs and correct positioning of the ribosomal proteins. One of the most intricate events that have recently been described is the removal of the ITS2-containing structure called pre-60S foot, which happens in a step-wise manner. Nop53 is an essential 60S AF that binds close to the ITS2 and plays a fundamental role in recruiting the RNA exosome for the 7S pre-rRNA processing, thereby dismantling the foot structure. Here we characterize the impact of Nop53 binding to the pre-60S on the compositional changes that happen during 60S assembly. For this purpose, preribosomes were affinity-purified with TAP-tagged 60S AFs (Nop7, Erb1, Rsa4, Arx1, Nmd3, Yvh1, and Lsg1) representative of different maturation stages both in the presence and absence of Nop53. Nop7 particles were also coimmunoprecipitated in the presence of Nop53 mutants incapable of recruiting the exosome (Nop53∆1-71, Nop53∆48-98) to compare with Nop53 depletion. The isolated preribosomes were analyzed by label-free MS/MS-based quantitative proteomics, revealing early and late-stage specific effects of Nop53 depletion.

2021-09-09 | PXD023383 | Pride

Targeted RNase H-mediated Extraction of crosslinked RBPs (TREX) for deciphering the region-specific interactome of 45S rRNA

Project description:We used Targeted RNase H-mediated Extraction of crosslinked RBPs (TREX)to assess the endogenous region-specific binding partners of 45S rRNA in human HCT116 cells. We performed TREX experiments against the full-length 45S, as well as each individual region (5'ETS. 18S, ITS1, 5.8S, ITS2, 28S, and 3'ETS). Extracted proteins from RNase H digested and control cells (4 or 5 replicate per region per condition) were compared, using label-free (LFQ) Quantitative proteomics.

2023-12-27 | PXD044659 | Pride

Pre-rRNA spatial distribution and functional organisation of the nucleolus

Project description:The multi-layered nucleolus serves as the primary site of ribosome biogenesis, where successive maturation of small (SSU) and large (LSU) ribosomal subunit precursors occur. However, the spatio-functional relationship between pre-rRNA processing and nucleolar substructures and how this adapts to changing cellular physiological demands have remained incompletely understood. Here, our spatiotemporal analyses revealed a compartment-specific ribosomal subunit processing in human nucleoli, with SSU processomes maintained in fibrillar center/dense fibrillar component/periphery dense fibrillar component (FC/DFC/PDFC) domains while LSU pre-rRNAs largely transited to PDFC/granular component (GC) regions. Slow proliferating cells exhibited unexpected 5' external transcribed space (5' ETS)-centered SSU processing impairment, accompanied by FC/DFC structural remodeling and retarded SSU outflux. Direct 5' ETS processing perturbation at least partially recapitulated these FC/DFC alterations, supporting the functional interdependence between SSU processing and nucleolar architecture. Notably, anamniote bipartite nucleoli with merged FC/DFC compartments, exhibited distinct 5' ETS distribution and slower pre-rRNA flux compared to multi-layered nucleoli in amniotes. Introducing a FC/DFC interface to bipartite nucleoli enhanced processing efficiency, indicating that evolutionary emergence of nested FC/DFC may have optimized pre-rRNA processing. Collectively, depicting the spatiotemporal distribution of pre-rRNAs revealed an essential role of 5' ETS-centered SSU processing in maintaining nucleolar substructures and suggested a possible evolutionary advantage of the multi-layered structure in amniotes.

2025-07-22 | GSE301874 | GEO

URB1 ensures 3' end processing of pre-rRNAs to prevent exosome-dependent degradation and is required for development

Project description:The nucleolus composes hundreds of proteins that play distinct roles in ribosomal RNA (rRNA) processing within Fibrillar Center/Dense Fibrillar Component (FC/DFC) units and ribosome assembly in Granular Component (GC). The sub-nucleolar localization of most proteins and how their unique localization facilitates rRNA processing have remained elusive. By screening 200 nucleolar candidate proteins with high-resolution, live-cell microscopy, we identified a previously undescribed sub-nucleolar compartment, named the periphery of DFC (PDFC). Among the 12 proteins identified in PDFC, URB1 (unhealthy ribosome biogenesis 1) is required for PDFC organization and proper 3' end processing of 47S pre-rRNA. URB1 binds between the 28S rRNA and the 3' external transcribed spacer (3' ETS) to ensure 3' ETS removal, which occurs at the DFC/PDFC boundary. Loss of URB1 leads to accumulation of aberrant 3' ETS-retained 32S pre-rRNA variants, which activates exosome-dependent nucleolar surveillance. This causes decreased 28S rRNA production, reduced cell proliferation and retarded mouse embryonic pre-implementation development. Furthermore, urb1 depletion results in developmental craniofacial disorder in zebrafish, which can be at least partially rescued by further depleting exosome components. Together, this study provides new insights into functional sub-nucleolar organizations, identifies a physiologically essential step in rRNA maturation and emphasizes the exosome-dependent pre-rRNA surveillance pathway.

2023-01-04 | GSE194413 | GEO

URB1 ensures 3' end processing of pre-rRNAs to prevent exosome-dependent degradation and is required for development [SHAPE-MaP]

2023-01-04 | GSE196625 | GEO

Coordinated control of mRNA and rRNA processing regulates early mammalian embryogenesis [ChIP-Seq_PolII]

Project description:Stem cells are regulated by transcriptional networks controlling pluripotency and differentiation. How basic cellular processes like splicing or protein synthesis regulate stem cell function is less well understood. Here, we show that the RNA binding protein HTATSF1 controls protein synthesis by controlling several independent RNA processing steps during ribosome biogenesis. In a complex with ribosomal RNA transcription and processing factors, HTATSF1 regulates ribosomal RNA abundance. By binding to the U2snRNP complex, HTATSF1 also controls intron removal specifically in ribosomal protein transcripts. HTATSF1-mediated control of protein synthesis is essential for the transition from the naïve pre-implantation epiblast to the primed post-implantation epiblast, a stage of low protein synthesis levels, and during further differentiation towards neuroectoderm. Our results identify coordinated regulation of ribosomal RNA and protein biosynthesis by HTATSF1 as an essential mechanism to mediate protein synthesis control during early stages of mammalian embryogenesis.

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Coordinated control of mRNA and rRNA processing regulates early mammalian embryogenesis [ChIP-Seq_HTATSF1]

2018-04-05 | GSE96593 | GEO

Coordinated control of mRNA and rRNA processing regulates early mammalian embryogenesis [RNA-Seq]

2018-04-05 | GSE96555 | GEO

The novel pre-rRNA detection workflow “Riboprobing” identified a hitherto undescribed aberrant pre-rRNA

Project description:Ribosomes translate mRNA into proteins and are essential for every living organism. In eukaryotes both ribosomal subunits are rapidly assembled in a strict hierarchical order, starting in the nucleolus with transcription of a common precursor ribosomal RNA (pre-rRNA). This pre-rRNA encodes three of the four mature rRNAs which are formed by several, consecutive endonucleolytic and exonucleolytic processing steps. Historically, Northern Blots are used to analyze the variety of different pre-rRNA species, only allowing rough length estimations. Although this limitation can be overcome with Primer Extension, both approaches often use radioactivity and are time consuming and costly. Here we present “Riboprobing” a reverse transcription-based workflow extended by linker ligation for easy and fast detection and characterization of various pre-rRNA species and their 5` as well as 3` ends. Using standard molecular biology lab equipment, our technique allows reliable discrimination of pre-rRNA species not resolved by Northern Blotting (e.g.: 27SA2, 27SA3 and 27SB). The method can be successfully used for analysis of total cell extracts as well as purified pre-ribosomes for a straightforward evaluation of the impact of mutant gene versions or inhibitors. In the course of method development, we identified and characterized a hitherto undescribed aberrant pre-rRNA, arising from LiCl inhibition. This pre-rRNA fragment spans from processing site A1 to E, forming a small RNP that is lacking most early joining assembly factors. This finding expands our knowledge of how the cell deals with severe pre-rRNA processing defects and demonstrates the strict requirement for the 5’ETS for the assembly process.

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