Interaction between the RNA-dependent ATPase and poly(A) polymerase subunits of the TRAMP complex is mediated by short peptides and important for snoRNA processing

Abstract

The RNA exosome is one of the main 3′ to 5′ exoribonucleases in eukaryotic cells. Although it is responsible for degradation or processing of a wide variety of substrate RNAs, it is very specific and distinguishes between substrate and non-substrate RNAs as well as between substrates that need to be 3′ processed and those that need to be completely degraded. This specificity does not appear to be determined by the exosome itself but rather by about a dozen other proteins. Four of these exosome cofactors have enzymatic activity, namely, the nuclear RNA-dependent ATPase Mtr4, its cytoplasmic paralog Ski2 and the nuclear non-canonical poly(A) polymerases, Trf4 and Trf5. Mtr4 and either Trf4 or Trf5 assemble into a TRAMP complex. However, how these enzymes assemble into a TRAMP complex and the functional consequences of TRAMP complex assembly remain unknown. Here, we identify an important interaction site between Mtr4 and Trf5, and show that disrupting the Mtr4/Trf interaction disrupts specific TRAMP and exosome functions, including snoRNA processing.

Substrate Specificity of the TRAMP Nuclear Surveillance Complexes

“…They were also less recovered with Mtr4Δarch relative to Mtr4, consistent with pre-rRNA maturation defects reported for strains carrying arch mutations (Fig. S1C) 13, 14, 38, 39 . In contrast, the major ncRNA classes, CUTs, SUTs and XUTs, were strongly recovered with Air2 and Trf4, relative to Air1 plus Trf5.…”

“…We compared strains in which the endogenous gene was HTP tagged for Air1, Air2, Trf4, Trf5, and Mtr4, as well as the exosome exonucleases Rrp44 and Rrp6. The Mtr4 arch domain is implicated in substrate recruitment 13, 14, 38, 39 , so we also constructed and analyzed a tagged Mtr4 mutant lacking this region (Mtr4Δarch) (Fig. S1A).…”

Gateway Arch to the RNA Exosome

“…Formation of the TRAMP complex requires the core helicase domains of the Mtr4, but not its arch domain (Falk et al, 2014;Losh et al, 2015). In addition to the interactions with the core helicase domains, a recent crystal structure shows the key arginine in the Mtr4 arch domain in contact with an aspartate on Air2 (Falk et al, 2014) that is also part of a conserved AIM-like motif (Figure 1).…”

Current perspectives on the role of TRAMP in nuclear RNA surveillance and quality control

“…48 A similar conserved region in the N-terminus of Trf5p is required for Mtr4p interaction. 3 The interactions between the subunits of the TRAMP complex are important for the coordination between helicase and polyadenylation activities of TRAMP. 43 On one hand, Trf4p with Air2p stimulates the RNA helicase activity of Mtr4p through increasing its ATP affinity.…”

“…1 In Saccharomyces cerevisiae, TRAMP complex is a heterotrimeric complex consisting of a poly(A) polymerase (either Trf4p or Trf5p); a zinc-knuckle ribonucleic acid (RNA)-binding protein (either Air1p or Air2p); and an RNA helicase Mtr4p. 2,3 The main function of TRAMP is to assist the nuclear exosome to degrade a large variety of RNA substrates, such as hypomodified initiator tRNA i Met , abnormally processed ribonucleic RNAs (rRNAs), [4][5][6] cryptic unstable transcripts (CUTs), [7][8][9][10] long noncoding RNAs (lncRNAs), 11 micro-RNAs (miRNAs), 12,13 and normal by-products of RNA metabolism such as spliced-out introns, 14,15 and to be involved in the many other RNA processes such as the maturation steps in precursor RNA processing and transfer RNA (tRNA) editing. 16 Therefore, it is not surprising that the TRAMP complex is also directly involved in many RNA processing pathways such as splicing, 15,17,18 RNA export, 19,20 and heterochromatic gene silencing, 21,22 and is even indirectly involved in the maintenance of genomic stability.…”