RNA Turnover: Unexpected Consequences of Being Tailed

In eukaryotic cells, the 3′ poly(A) tails found on mRNA influence their stability and translation. The discovery of a second nuclear poly(A) polymerase complex has fueled a series of reports defining a new and unexpected role for 3′ end poly(A) tails in the nuclear surveillance and turnover of noncoding RNAs and intergenic mRNAs of unknown function

Loss of The RNA Helicase SKIV2L2 Impairs Mitotic Progression and Replication-Dependent Histone mRNA Turnover in Murine Cell Lines

RNA surveillance via the nuclear exosome requires cofactors such as the helicase SKIV2L2 to process and degrade certain noncoding RNAs. This research aimed to characterize the phenotype associated with RNAi knockdown of Skiv2l2 in two murine cancer cell lines: Neuro2A and P19. SKIV2L2 depletion in Neuro2A and P19 cells induced changes in gene expression indicative of cell differentiation and reduced cellular proliferation by 30%. Propidium iodide-based cell-cycle analysis of Skiv2l2 knockdown cells revealed defective progression through the G2/M phase and an accumulation of mitotic cells, suggesting SKIV2L2 contributes to mitotic progression. Since SKIV2L2 targets RNAs to the nuclear exosome for processing and degradation, we identified RNA targets elevated in cells depleted of SKIV2L2 that could account for the observed twofold increase in mitotic cells. Skiv2l2knockdown cells accumulated replication-dependent histone mRNAs, among other RNAs, that could impede mitotic progression and indirectly trigger differentiation

Mutations in Mtr4 Structural Domains Reveal Their Important Role in Regulating tRNA\u3csub\u3ei\u3c/sub\u3e \u3csup\u3eMet\u3c/sup\u3e Turnover in \u3cem\u3eSaccharomyces cerevisiae\u3c/em\u3e and Mtr4p Enzymatic Activities \u3cem\u3eIn Vitro\u3c/em\u3e

RNA processing and turnover play important roles in the maturation, metabolism and quality control of a large variety of RNAs thereby contributing to gene expression and cellular health. The TRAMP complex, composed of Air2p, Trf4p and Mtr4p, stimulates nuclear exosome-dependent RNA processing and degradation in Saccharomyces cerevisiae. The Mtr4 protein structure is composed of a helicase core and a novel so-called arch domain, which protrudes from the core. The helicase core contains highly conserved helicase domains RecA-1 and 2, and two structural domains of unclear functions, winged helix domain (WH) and ratchet domain. How the structural domains (arch, WH and ratchet domain) coordinate with the helicase domains and what roles they are playing in regulating Mtr4p helicase activity are unknown. We created a library of Mtr4p structural domain mutants for the first time and screened for those defective in the turnover of TRAMP and exosome substrate, hypomodified tRNAiMet. We found these domains regulate Mtr4p enzymatic activities differently through characterizing the arch domain mutants K700N and P731S, WH mutant K904N, and ratchet domain mutant R1030G. Arch domain mutants greatly reduced Mtr4p RNA binding, which surprisingly did not lead to significant defects on either in vivo tRNAiMet turnover, or in vitro unwinding activities. WH mutant K904N and Ratchet domain mutant R1030G showed decreased tRNAiMet turnover in vivo, as well as reduced RNA binding, ATPase and unwinding activities of Mtr4p in vitro. Particularly, K904 was found to be very important for steady protein levels in vivo. Overall, we conclude that arch domain plays a role in RNA binding but is largely dispensable for Mtr4p enzymatic activities, however the structural domains in the helicase core significantly contribute to Mtr4p ATPase and unwinding activities

The Exosome Subunit Rrp44 Plays a Direct Role in RNA Substrate Recognition

The exosome plays key roles in RNA maturation and surveillance, but it is unclear how target RNAs are identified. We report the functional characterization of the yeast exosome component Rrp44, a member of the RNase II family. Recombinant Rrp44 and the purified TRAMP polyadenylation complex each specifically recognized tRNAiMet lacking a single m1A58 modification, even in the presence of a large excess of total tRNA. This tRNA is otherwise mature and functional in translation in vivo but is presumably subtly misfolded. Complete degradation of the hypomodified tRNA required both Rrp44 and the poly(A) polymerase activity of TRAMP. The intact exosome lacking only the catalytic activity of Rrp44 failed to degrade tRNAiMet, showing this to be a specific Rrp44 substrate. Recognition of hypomodified tRNAiMet by Rrp44 is genetically separable from its catalytic activity on other substrates, with the mutations mapping to distinct regions of the protein

Nuclear RNA Surveillance in \u3cem\u3eSaccharomyces cerevisiae\u3c/em\u3e: Trf4p-dependent Polyadenylation of Nascent Hypomethylated tRNA and an Aberrant Form of 5S rRNA

1-Methyladenosine modification at position 58 of tRNA is catalyzed by a two-subunit methyltransferase composed of Trm6p and Trm61p in Saccharomyces cerevisiae. Initiator tRNA (tRNAiMet) lacking m1A58 (hypomethylated) is rendered unstable through the cooperative function of the poly(A) polymerases, Trf4p/Trf5p, and the nuclear exosome. We provide evidence that a catalytically active Trf4p poly(A) polymerase is required for polyadenylation of hypomethylated tRNAiMet in vivo. DNA sequence analysis of tRNAiMet cDNAs and Northern hybridizations of poly(A)+ RNA provide evidence that nascent pre-tRNAiMet transcripts are targeted for polyadenylation and degradation. We determined that a mutant U6 snRNA and an aberrant form of 5S rRNA are stabilized in the absence of Trf4p, supporting that Trf4p facilitated RNA surveillance is a global process that stretches beyond hypomethylated tRNAiMet. We conclude that an array of RNA polymerase III transcripts are targeted for Trf4p/ Trf5p-dependent polyadenylation and turnover to eliminate mutant and variant forms of normally stable RNAs

RNA Unwinding by the Trf4/Air2/Mtr4 Polyadenylation (TRAMP) Complex

Many RNA-processing events in the cell nucleus involve the Trf4/Air2/Mtr4 polyadenylation (TRAMP) complex, which contains the poly(A) polymerase Trf4p, the Zn-knuckle protein Air2p, and the RNA helicase Mtr4p. TRAMP polyadenylates RNAs designated for processing by the nuclear exosome. In addition, TRAMP functions as an exosome cofactor during RNA degradation, and it has been speculated that this role involves disruption of RNA secondary structure. However, it is unknown whether TRAMP displays RNA unwinding activity. It is also not clear how unwinding would be coordinated with polyadenylation and the function of the RNA helicase Mtr4p in modulating poly(A) addition. Here, we show that TRAMP robustly unwinds RNA duplexes. The unwinding activity of Mtr4p is significantly stimulated by Trf4p/Air2p, but the stimulation of Mtr4p does not depend on ongoing polyadenylation. Nonetheless, polyadenylation enables TRAMP to unwind RNA substrates that it otherwise cannot separate. Moreover, TRAMP displays optimal unwinding activity on substrates with a minimal Mtr4p binding site comprised of adenylates. Our results suggest a model for coordination between unwinding and polyadenylation activities by TRAMP that reveals remarkable synergy between helicase and poly(A) polymerase

Degradation of Hypomodified tRNA\u3csub\u3ei\u3c/sub\u3e\u3csup\u3eMet\u3c/sup\u3e in vivo Involves RNA-dependent ATPase Activity of the DExH Helicase Mtr4p

Effective turnover of many incorrectly processed RNAs in yeast, including hypomodified tRNAi Met, requires the TRAMP complex, which appends a short poly(A) tail to RNA designated for decay. The poly(A) tail stimulates degradation by the exosome. The TRAMP complex contains the poly(A) polymerase Trf4p, the RNA-binding protein Air2p, and the DExH RNA helicase Mtr4p. The role of Mtr4p in RNA degradation processes involving the TRAMP complex has been unclear. Here we show through a genetic analysis that MTR4 is required for degradation but not for polyadenylation of hypomodified tRNAi Met. A suppressor of the trm6-504 mutation in the tRNA m1A58 methyltransferase (Trm6p/Trm61p), which causes a reduced level of tRNAi Met, was mapped to MTR4. This mtr4-20 mutation changed a single amino acid in the conserved helicase motif VI of Mtr4p. The mutation stabilizes hypomodified tRNAi Met in vivo but has no effect on TRAMP complex stability or polyadenylation activity in vivo or in vitro. We further show that purified recombinant Mtr4p displays RNA-dependent ATPase activity and unwinds RNA duplexes with a 3′-to-5′ polarity in an ATP-dependent fashion. Unwinding and RNA-stimulated ATPase activities are strongly reduced in the recombinant mutant Mtr4-20p, suggesting that these activities of Mtr4p are critical for degradation of polyadenylated hypomodified tRNAi Met

Unexpected Accumulation of ncm\u3csup\u3e5\u3c/sup\u3eU and ncm\u3csup\u3e5\u3c/sup\u3es\u3csup\u3e2\u3c/sup\u3eU in a \u3cem\u3etrm9\u3c/em\u3e Mutant Suggests an Additional Step in the Synthesis of mcm\u3csup\u3e5\u3c/sup\u3eU and mcm\u3csup\u3e5\u3c/sup\u3es\u3csup\u3e2\u3c/sup\u3eU

Background Transfer RNAs are synthesized as a primary transcript that is processed to produce a mature tRNA. As part of the maturation process, a subset of the nucleosides are modified. Modifications in the anticodon region often modulate the decoding ability of the tRNA. At position 34, the majority of yeast cytosolic tRNA species that have a uridine are modified to 5-carbamoylmethyluridine (ncm5U), 5-carbamoylmethyl-2′-O-methyluridine (ncm5Um), 5-methoxycarbonylmethyl-uridine (mcm5U) or 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U). The formation of mcm5 and ncm5 side chains involves a complex pathway, where the last step in formation of mcm5 is a methyl esterification of cm5 dependent on the Trm9 and Trm112 proteins. Methodology and Principal Findings Both Trm9 and Trm112 are required for the last step in formation of mcm5 side chains at wobble uridines. By co-expressing a histidine-tagged Trm9p together with a native Trm112p in E. coli, these two proteins purified as a complex. The presence of Trm112p dramatically improves the methyltransferase activity of Trm9p in vitro. Single tRNA species that normally contain mcm5U or mcm5s2U nucleosides were isolated from trm9Δ or trm112Δ mutants and the presence of modified nucleosides was analyzed by HPLC. In both mutants, mcm5U and mcm5s2U nucleosides are absent in tRNAs and the major intermediates accumulating were ncm5U and ncm5s2U, not the expected cm5U and cm5s2U. Conclusions Trm9p and Trm112p function together at the final step in formation of mcm5U in tRNA by using the intermediate cm5U as a substrate. In tRNA isolated from trm9Δ and trm112Δ strains, ncm5U and ncm5s2U nucleosides accumulate, questioning the order of nucleoside intermediate formation of the mcm5 side chain. We propose two alternative explanations for this observation. One is that the intermediate cm5U is generated from ncm5U by a yet unknown mechanism and the other is that cm5U is formed before ncm5U and mcm5U

An Invertible Linearization Map for the Quartic Oscillator

The set of world lines for the non-relativistic quartic oscillator satisfying Newton's equation of motion for all space and time in 1-1 dimensions with no constraints other than the "spring" restoring force is shown to be equivalent (1-1-onto) to the corresponding set for the harmonic oscillator. This is established via an energy preserving invertible linearization map which consists of an explicit nonlinear algebraic deformation of coordinates and a nonlinear deformation of time coordinates involving a quadrature. In the context stated, the map also explicitly solves Newton's equation for the quartic oscillator for arbitrary initial data on the real line. This map is extended to all attractive potentials given by even powers of the space coordinate. It thus provides classes of new solutions to the initial value problem for all these potentials

The Bipartite Structure of the tRNA m\u3csup\u3e1\u3c/sup\u3eA58 Methyltransferase from \u3cem\u3eS. cerevisiae\u3c/em\u3e is Conserved in Humans

Among all types of RNA, tRNA is unique given that it possesses the largest assortment and abundance of modified nucleosides. The methylation at N1 of adenosine 58 is a conserved modification, occurring in bacterial, archaeal, and eukaryotic tRNAs. In the yeast Saccharomyces cerevisiae, the tRNA 1-methyladenosine 58 (m1A58) methyltransferase (Mtase) is a two-subunit enzyme encoded by the essential genes TRM6 (GCD10) and TRM61 (GCD14). While the significance of many tRNA modifications is poorly understood, methylation of A58 is known to be critical for maintaining the stability of initiator tRNAMet in yeast. Furthermore, all retroviruses utilize m1A58-containing tRNAs to prime reverse transcription, and it has been shown that the presence of m1A58 in human tRNA3 Lys is needed for accurate termination of plus-strand strong-stop DNA synthesis during HIV-1 replication. In this study we have identified the human homologs of the yeast m1A Mtase through amino acid sequence identity and complementation of trm6 and trm61 mutant phenotypes. When coexpressed in yeast, human Trm6p and Trm61p restored the formation of m1A in tRNA, modifying both yeast initiator tRNAMet and human tRNA3 Lys. Stable hTrm6p/hTrm61p complexes purified from yeast maintained tRNA m1A Mtase activity in vitro. The human m1A Mtase complex also exhibited substrate specificity—modifying wild-type yeast tRNAi Met but not an A58U mutant. Therefore, the human tRNA m1A Mtase shares both functional and structural homology with the yeast tRNA m1A Mtase, possessing similar enzymatic activity as well as a conserved binary composition