mRNAs containing the histone 3' stem-loop are degraded primarily by decapping mediated by oligouridylation of the 3' end

RNA. 2013 Jan;19(1):1-16. doi: 10.1261/rna.034470.112. Epub 2012 Nov 27.

Abstract

Metazoan replication-dependent histone mRNAs are only present in S-phase, due partly to changes in their stability. These mRNAs end in a unique stem-loop (SL) that is required for both translation and cell-cycle regulation. Previous studies showed that histone mRNA degradation occurs through both 5'→3' and 3'→5' processes, but the relative contributions are not known. The 3' end of histone mRNA is oligouridylated during its degradation, although it is not known whether this is an essential step. We introduced firefly luciferase reporter mRNAs containing the histone 3' UTR SL (Luc-SL) and either a normal or hDcp2-resistant cap into S-phase HeLa cells. Both mRNAs were translated, and translation initially protected the mRNAs from degradation, but there was a lag of ∼40 min with the uncleavable cap compared to ∼8 min for the normal cap before rapid decay. Knockdown of hDcp2 resulted in a similar longer lag for Luc-SL containing a normal cap, indicating that 5'→3' decay is important in this system. Inhibition of DNA replication with hydroxyurea accelerated the degradation of Luc-SL. Knockdown of terminal uridyltransferase (TUTase) 4 but not TUTase 3 slowed the decay process, but TUTase 4 knockdown had no effect on destabilization of the mRNA by hydroxyurea. Both Luc-SL and its 5' decay intermediates were oligouridylated. Preventing oligouridylation by 3'-deoxyadenosine (cordycepin) addition to the mRNA slowed degradation, in the presence or absence of hydroxyurea, suggesting oligouridylation initiates degradation. The spectrum of oligouridylated fragments suggests the 3'→5' degradation machinery stalls during initial degradation, whereupon reuridylation occurs.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3' Untranslated Regions / physiology
  • DNA Replication / drug effects
  • Deoxyadenosines / pharmacology
  • Gene Silencing
  • HeLa Cells
  • Histones / metabolism
  • Humans
  • Hydroxyurea / pharmacology
  • Nucleic Acid Synthesis Inhibitors / pharmacology
  • Oligoribonucleotides / antagonists & inhibitors
  • Oligoribonucleotides / metabolism*
  • Polynucleotide Adenylyltransferase
  • Protein Biosynthesis
  • RNA Stability / genetics
  • RNA Stability / physiology*
  • RNA, Messenger / chemistry
  • RNA, Messenger / metabolism*
  • Transduction, Genetic
  • Uracil Nucleotides / antagonists & inhibitors
  • Uracil Nucleotides / metabolism*
  • mRNA Cleavage and Polyadenylation Factors

Substances

  • 3' Untranslated Regions
  • Deoxyadenosines
  • Histones
  • Nucleic Acid Synthesis Inhibitors
  • Oligoribonucleotides
  • RNA, Messenger
  • Uracil Nucleotides
  • mRNA Cleavage and Polyadenylation Factors
  • oligo(U)
  • Polynucleotide Adenylyltransferase
  • TENT2 protein, human
  • cordycepin
  • Hydroxyurea