Mature ribosomal RNAs (rRNAs) are produced from polycistronic precursors following complex processing. Precursor (pre)-rRNA processing has been extensively characterized in yeast and was assumed to be conserved in humans. We functionally characterized 625 nucleolar proteins in HeLa cells and identified 286 required for processing, including 74 without a yeast homolog. For selected candidates, we demonstrated that pre-rRNA processing defects are conserved in different cell types (including primary cells), defects are not due to activation of a p53-dependent nucleolar tumor surveillance pathway, and they precede cell-cycle arrest and apoptosis. We also investigated the exosome's role in processing internal transcribed spacers (ITSs) and report that 3' end maturation of 18S rRNA involves EXOSC10/Rrp6, a yeast ITS2 processing factor. We conclude that human cells adopt unique strategies and recruit distinct trans-acting factors to carry out essential processing steps, posing fundamental implications for understanding ribosomopathies at the molecular level and developing effective therapeutic agents.
A group II intron encoding a protein belonging to the LAGLIDADG family of homing endonucleases was identified in the mitochondrial rns gene of the filamentous fungus Leptographium truncatum, and the catalytic activities of both the intron and its encoded protein were characterized. A model of the RNA secondary structure indicates that the intron is a member of the IIB1 subclass and the open reading frame is inserted in ribozyme domain III. In vitro assays carried out with two versions of the intron, one in which the open reading frame was removed and the other in which it was present, demonstrate that both versions of the intron readily self-splice at 37°C and at a concentration of MgCl 2 as low as 6 mM. The open reading frame encodes a functional LAGLIDADG homing endonuclease that cleaves 2 (top strand) and 6 (bottom strand) nucleotides (nt) upstream of the intron insertion site, generating 4 nt 39 OH overhangs. In vitro splicing assays carried out in the absence and presence of the intron-encoded protein indicate that the protein does not enhance intron splicing, and RNA-binding assays show that the protein does not appear to bind to the intron RNA precursor transcript. These findings raise intriguing questions concerning the functional and evolutionary relationships of the two components of this unique composite element.
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