The roles of SSU processome components and surveillance factors in the initial processing of human ribosomal RNA

RNA. 2014 Apr;20(4):540-50. doi: 10.1261/rna.043471.113. Epub 2014 Feb 18.

Abstract

During eukaryotic ribosome biogenesis, three of the mature ribosomal (r)RNAs are released from a single precursor transcript (pre-rRNA) by an ordered series of endonucleolytic cleavages and exonucleolytic processing steps. Production of the 18S rRNA requires the removal of the 5' external transcribed spacer (5'ETS) by endonucleolytic cleavages at sites A0 and A1/site 1. In metazoans, an additional cleavage in the 5'ETS, at site A', upstream of A0, has also been reported. Here, we have investigated how A' processing is coordinated with assembly of the early preribosomal complex. We find that only the tUTP (UTP-A) complex is critical for A' cleavage, while components of the bUTP (UTP-B) and U3 snoRNP are important, but not essential, for efficient processing at this site. All other factors involved in the early stages of 18S rRNA processing that were tested here function downstream from this processing step. Interestingly, we show that the RNA surveillance factors XRN2 and MTR4 are also involved in A' cleavage in humans. A' cleavage is largely bypassed when XRN2 is depleted, and we also discover that A' cleavage is not always the initial processing event in all cell types. Together, our data suggest that A' cleavage is not a prerequisite for downstream pre-rRNA processing steps and may, in fact, represent a quality control step for initial pre-rRNA transcripts. Furthermore, we show that components of the RNA surveillance machinery, including the exosome and TRAMP complexes, also play key roles in the recycling of excised spacer fragments and degradation of aberrant pre-rRNAs in human cells.

Keywords: nuclease; nucleolus; rRNA processing; ribosome; snoRNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Northern
  • Exoribonucleases / genetics
  • Exoribonucleases / metabolism*
  • Exosomes / metabolism
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • MCF-7 Cells
  • RNA Helicases / genetics
  • RNA Helicases / metabolism*
  • RNA Precursors / genetics*
  • RNA Precursors / metabolism
  • RNA Processing, Post-Transcriptional*
  • RNA, Messenger / genetics
  • RNA, Ribosomal / genetics*
  • RNA, Ribosomal / metabolism
  • Real-Time Polymerase Chain Reaction
  • Reverse Transcriptase Polymerase Chain Reaction
  • Ribonucleoproteins, Small Nucleolar / genetics
  • Ribonucleoproteins, Small Nucleolar / metabolism
  • Ribosome Subunits, Small / metabolism*

Substances

  • RNA Precursors
  • RNA, Messenger
  • RNA, Ribosomal
  • Ribonucleoproteins, Small Nucleolar
  • Exoribonucleases
  • XRN2 protein, human
  • SKIV2L2 protein, human
  • RNA Helicases