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

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 59/39 and 39/59 processes, but the relative contributions are not known. The 39 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 39 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 59/39 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 59 decay intermediates were oligouridylated. Preventing oligouridylation by 39-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 39/59 degradation machinery stalls during initial degradation, whereupon reuridylation occurs.

The C-terminal extension of Lsm4 interacts directly with the 3′ end of the histone mRNP and is required for efficient histone mRNA degradation

“…The cytoplasmic histone mRNA ends 2-3 nt after the stemloop as a result of exonucleolytic trimming by the 3 ′ hExo (Hoefig et al 2013), which forms a stable ternary complex (SLBP:SL:3 ′ hExo) with SLBP and the stem-loop (Yang et al 2006;Tan et al 2013). The initial intermediate in histone mRNA degradation is oligouridylation of the 3 ′ end of histone mRNA (Su et al 2013). Much of the histone mRNA is degraded 3 ′ -5, since knockdown of the exosome has a much greater effect on histone mRNA degradation than knockdown of Dcp2 or Xrn1 (Mullen and Marzluff 2008).…”

“…A fraction of the histone mRNAs is certainly degraded by the 5 ′ -3 ′ degradation pathway (Mullen and Marzluff 2008;Su et al 2013). However, after inhibition of DNA replication, there is an increase in the fraction of the histone mRNAs that is degraded by the 3 ′ -5 ′ pathway (Su et al 2013), requiring the exosome (Mullen and Marzluff 2008).…”

“…However, after inhibition of DNA replication, there is an increase in the fraction of the histone mRNAs that is degraded by the 3 ′ -5 ′ pathway (Su et al 2013), requiring the exosome (Mullen and Marzluff 2008). It seems likely that removal of the oligo(U) tail either by 3 ′ hExo or the exosome is initiated with Lsm1-7 bound to the tail, and indeed it is possible that Lsm1-7 plays a direct role in removal of the Utail rather than simply protecting the tail from degradation.…”

“…Histone mRNA degradation requires that the histone mRNA be actively translated and requires the nonsense-mediated decay (NMD) factor Upf1, suggesting that translation termination may become inefficient as part of triggering initiation of histone mRNA degradation (Kaygun and Marzluff 2005a,b). The initial biochemical step in degradation is covalent alteration of the histone mRNA by addition of an oligo(U) tail to the 3 ′ end (Mullen and Marzluff 2008;Su et al 2013). Knockdown of Lsm1 inhibits degradation of histone mRNA consistent with Lsm1-7 being recruited to the oligo(U) tail.…”

Oligoadenylation of 3′ decay intermediates promotes cytoplasmic mRNA degradation in Drosophila cells

“…Consistent with a role in 3 ′ decay, U residues are added not only to poly(A) tails but also to the upstream fragments generated by siRNAdirected mRNA cleavage in Arabidopsis and mouse cells (Shen and Goodman 2004) and to 3 ′ decay intermediates of mammalian histone mRNAs (Mullen and Marzluff 2008;Hoefig et al 2013;Slevin et al 2014). Like Trf4p and Trf5p, the enzymes responsible for the addition of U residues are members of the enzyme family of poly(U) polymerases or terminal uridilyl transferases (TUTases) and noncanonical poly(A) polymerases (Kwak and Wickens 2007;Martin and Keller 2007;Rissland et al 2007;Rissland and Norbury 2009;Morozov et al 2010Morozov et al , 2012Schmidt and Norbury 2010;Schmidt et al 2011;Su et al 2013;Lim et al 2014).…”