Substrate Specificity of the TRAMP Nuclear Surveillance Complexes

Abstract

During nuclear surveillance in yeast, the RNA exosome functions together with the TRAMP complexes. These include the DEAH-box RNA helicase Mtr4 together with an RNA-binding protein (Air1 or Air2) and a poly(A) polymerase (Trf4 or Trf5). To better determine how RNA substrates are targeted, we analyzed protein and RNA interactions for TRAMP components. Mass spectrometry identified three distinct TRAMP complexes formed in vivo. These complexes preferentially assemble on different classes of transcripts. Unexpectedly, on many substrates, including pre-rRNAs and pre-mRNAs, binding specificity was apparently conferred by Trf4 and Trf5. Clustering of mRNAs by TRAMP association showed co-enrichment for mRNAs with functionally related products, supporting the significance of surveillance in regulating gene expression. We compared binding sites of TRAMP components with multiple nuclear RNA binding proteins, revealing preferential colocalization of subsets of factors. TRF5 deletion reduced Mtr4 recruitment and increased RNA abundance for mRNAs specifically showing high Trf5 binding.

System-wide analyses of the fission yeast poly(A)+ RNA interactome reveal insights into organisation and function of RNA-protein complexes

“…RNA can also be degraded by Rrp6 exonuclease that is located on the top of the exosome. In addition, it has been proposed that RNA can access Dis3 directly without passing through the channel ("direct access", da conformation) (Bonneau et al, 2009;Delan-Forino et al, 2017;Han and van Hoof, 2016;Liu et al, 2014;Makino et al, 2013Makino et al, , 2015. However, the functional contribution of each model as well the mechanisms that direct routing of substrates have remain unexplored.…”

“…Behaviour of a given protein in the RIC experiment reflects the mean RNA association of this protein in an ensemble of states. It was suggested that in wild-type, the preferred state of the nuclear exosome is the actively channel-threading conformation (Bonneau et al, 2009;Delan-Forino et al, 2017;Schneider et al, 2012). To test this hypothesis, we sought to perturb the system by using defined mutants of the exosome complex.…”

“…Hence, the positive shift of the Dis3/Rrp43 signature in the comparative RIC experiment is compatible with an increased use of the da route by poly(A)+ RNA in a rrp6Δ mutant ( Figure 7C). So far, the da conformation has only been observed in S. cerevisiae, and was suggested to prefer short structured substrates, such as pre-tRNAs and hypomodified tRNA (Delan-Forino et al, 2017;Han and van Hoof, 2016). However, an S. cerevisiae mutant that is unable to adopt the da conformationand that has no discernible growth phenotype in a wild-type backgroundshows a synthetic growth defect with rrp6Δ (Han and van Hoof, 2016).…”

“…Adaptation of the da conformation could explain how functional redundancy between Dis3 and Rrp6 is achieved to allow efficient regulation of cellular RNA levels. This would enable Dis3 to execute degradation of Rrp6 substrates in its absence (Delan-Forino et al, 2017).…”

Substrate discrimination and quality control require each catalytic activity of TRAMP and the nuclear RNA exosome

“…Budding yeast TRAMP includes one of two nontemplated poly(A) polymerases of the Polβ family, Trf4 or Trf5, and one of two zinc-knuckle RNA-binding proteins, Air1 or Air2 (24)(25)(26)(27). TRAMP promotes degradation of RNA transcripts produced by all three RNA polymerases (28) through the addition of short oligo-adenylated tails to the 3′ end of RNAs (24,25,29,30).…”