Studies of the 5' exonuclease and endonuclease activities of CPSF-73 in histone pre-mRNA processing

Mol Cell Biol. 2009 Jan;29(1):31-42. doi: 10.1128/MCB.00776-08. Epub 2008 Oct 27.

Abstract

Processing of histone pre-mRNA requires a single 3' endonucleolytic cleavage guided by the U7 snRNP that binds downstream of the cleavage site. Following cleavage, the downstream cleavage product (DCP) is rapidly degraded in vitro by a nuclease that also depends on the U7 snRNP. Our previous studies demonstrated that the endonucleolytic cleavage is catalyzed by the cleavage/polyadenylation factor CPSF-73. Here, by using RNA substrates with different nucleotide modifications, we characterize the activity that degrades the DCP. We show that the degradation is blocked by a 2'-O-methyl nucleotide and occurs in the 5'-to-3' direction. The U7-dependent 5' exonuclease activity is processive and continues degrading the DCP substrate even after complete removal of the U7-binding site. Thus, U7 snRNP is required only to initiate the degradation. UV cross-linking studies demonstrate that the DCP and its 5'-truncated version specifically interact with CPSF-73, strongly suggesting that in vitro, the same protein is responsible for the endonucleolytic cleavage of histone pre-mRNA and the subsequent degradation of the DCP. By using various RNA substrates, we define important space requirements upstream and downstream of the cleavage site that dictate whether CPSF-73 functions as an endonuclease or a 5' exonuclease. RNA interference experiments with HeLa cells indicate that degradation of the DCP does not depend on the Xrn2 5' exonuclease, suggesting that CPSF-73 degrades the DCP both in vitro and in vivo.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Base Sequence
  • Cleavage And Polyadenylation Specificity Factor / metabolism*
  • Drosophila
  • Endonucleases / metabolism*
  • Exoribonucleases / metabolism*
  • Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / metabolism
  • HeLa Cells
  • Histones / metabolism*
  • Humans
  • Mice
  • Molecular Sequence Data
  • Nucleotides / metabolism
  • Phosphates / metabolism
  • Phosphorus Radioisotopes
  • RNA Precursors / chemistry
  • RNA Precursors / metabolism*
  • RNA Processing, Post-Transcriptional*
  • RNA Stability
  • Regulatory Sequences, Nucleic Acid / genetics
  • Ribonucleoprotein, U7 Small Nuclear / metabolism
  • Substrate Specificity

Substances

  • Cleavage And Polyadenylation Specificity Factor
  • Histones
  • Nucleotides
  • Phosphates
  • Phosphorus Radioisotopes
  • RNA Precursors
  • Ribonucleoprotein, U7 Small Nuclear
  • Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)
  • Endonucleases
  • Exoribonucleases
  • XRN2 protein, human