Aneeshkumar G. Arimbasseri

Mechanism of Transcription Termination by RNA Polymerase III Utilizes a Non-template Strand Sequence-Specific Signal Element

Understanding the mechanism of transcription termination by a eukaryotic RNA polymerase (RNAP) has been limited by lack of a characterizable intermediate that reflects transition from an elongation complex to a true termination event. While other multisubunit RNAPs require multipartite cis-signals and/or ancillary factors to mediate pausing and release of the nascent transcript from the clutches of these enzymes, RNAP III does so with precision and efficiency on a simple oligo(dT) tract, independent of other cis-elements or trans-factors. We report an RNAP III pre-termination complex that reveals termination mechanisms controlled by sequence-specific elements in the non-template strand. Furthermore, the TFIIF-like RNAP III subunit C37 is required for this function of the non-template strand signal. The results reveal the RNAP III terminator as an information-rich control element. While the template strand promotes destabilization via a weak oligo(rU:dA) hybrid, the non-template strand provides distinct sequence-specific destabilizing information through interactions with the C37 subunit.

Comparative overview of RNA polymerase II and III transcription cycles, with focus on RNA polymerase III termination and reinitiation

In eukaryotes, RNA polymerase (RNAP) III transcribes hundreds of genes for tRNAs and 5S rRNA, among others, which share similar promoters and stable transcription initiation complexes (TIC), which support rapid RNAP III recycling. In contrast, RNAP II transcribes a large number of genes with highly variable promoters and interacting factors, which exert fine regulatory control over TIC lability and modifications of RNAP II at different transitional points in the transcription cycle. We review data that illustrate a relatively smooth continuity of RNAP III initiation-elongation-termination and reinitiation toward its function to produce high levels of tRNAs and other RNAs that support growth and development.

RNA Polymerase III Output Is Functionally Linked to tRNA Dimethyl-G26 Modification.

Control of the differential abundance or activity of tRNAs can be important determinants of gene regulation. RNA polymerase (RNAP) III synthesizes all tRNAs in eukaryotes and it derepression is associated with cancer. Maf1 is a conserved general repressor of RNAP III under the control of the target of rapamycin (TOR) that acts to integrate transcriptional output and protein synthetic demand toward metabolic economy. Studies in budding yeast have indicated that the global tRNA gene activation that occurs with derepression of RNAP III via maf1-deletion is accompanied by a paradoxical loss of tRNA-mediated nonsense suppressor activity, manifested as an antisuppression phenotype, by an unknown mechanism. We show that maf1-antisuppression also occurs in the fission yeast S. pombe amidst general activation of RNAP III. We used tRNA-HydroSeq to document that little changes occurred in the relative levels of different tRNAs in maf1Δ cells. By contrast, the efficiency of N2,N2-dimethyl G26 (m2 2G26) modification on certain tRNAs was decreased in response to maf1-deletion and associated with antisuppression, and was validated by other methods. Over-expression of Trm1, which produces m2 2G26, reversed maf1-antisuppression. A model that emerges is that competition by increased tRNA levels in maf1Δ cells leads to m2 2G26 hypomodification due to limiting Trm1, reducing the activity of suppressor-tRNASerUCA and accounting for antisuppression. Consistent with this, we show that RNAP III mutations associated with hypomyelinating leukodystrophy decrease tRNA transcription, increase m2 2G26 efficiency and reverse antisuppression. Extending this more broadly, we show that a decrease in tRNA synthesis by treatment with rapamycin leads to increased m2 2G26 modification and that this response is conserved among highly divergent yeasts and human cells.

Chromatin Structure and Expression of a Gene Transcribed by RNA Polymerase III Are Independent of H2A.Z Deposition

ABSTRACT The genes transcribed by RNA polymerase III (Pol III) generally have intragenic promoter elements. One of them, the yeast U6 snRNA (SNR6) gene is activated in vitro by a positioned nucleosome between its intragenic box A and extragenic, downstream box B separated by ∼200 bp. We demonstrate here that the in vivo chromatin structure of the gene region is characterized by the presence of an array of positioned nucleosomes, with only one of them in the 5′ end of the gene having a regulatory role. A positioned nucleosome present between boxes A and B in vivo does not move when the gene is repressed due to nutritional deprivation. In contrast, the upstream nucleosome which covers the TATA box under repressed conditions is shifted ∼50 bp further upstream by the ATP-dependent chromatin remodeler RSC upon activation. It is marked with the histone variant H2A.Z and H4K16 acetylation in active state. In the absence of H2A.Z, the chromatin structure of the gene does not change, suggesting that H2A.Z is not required for establishing the active chromatin structure. These results show that the chromatin structure directly participates in regulation of a Pol III-transcribed gene under different states of its activity in vivo.

Point mutations in the Rpb9-homologous domain of Rpc11 that impair transcription termination by RNA polymerase III

RNA polymerase III recognizes and pauses at its terminator, an oligo(dT) tract in non-template DNA, terminates 3′ oligo(rU) synthesis within this sequence, and releases the RNA. The pol III subunit Rpc11p (C11) mediates RNA 3′–5′ cleavage in the catalytic center of pol III during pausing. The amino and carboxyl regions of C11 are homologous to domains of the pol II subunit Rpb9p, and the pol II elongation and RNA cleavage factor, TFIIS, respectively. We isolated C11 mutants from Schizosaccharomyces pombe that cause pol III to readthrough terminators in vivo. Mutant RNA confirmed the presence of terminator readthrough transcripts. A predominant mutation site, F32, resides in the C11 Rpb9-like domain. Another mutagenic approach confirmed the F32 mutation and also isolated I34 and Y30 mutants. Modeling Y30, F32 and I34 of C11 in available cryoEM pol III structures predicts a hydrophobic patch that may interface with C53/37. Another termination mutant, Rpc2-T455I, appears to reside internally, near the RNA–DNA hybrid. We show that the Rpb9 and TFIIS homologous mutants of C11 reflect distinct activities, that differentially affect terminator recognition and RNA 3′ cleavage. We propose that these C11 domains integrate action at the upper jaw and center of pol III during termination.

RNA Polymerase III Advances: Structural and tRNA Functional Views

RNA synthesis in eukaryotes is divided among three RNA polymerases (RNAPs). RNAP III transcribes hundreds of tRNA genes and fewer additional short RNA genes. We survey recent work on transcription by RNAP III including an atomic structure, mechanisms of action, interactions with chromatin and retroposons, and a conserved link between its activity and a tRNA modification that enhances mRNA decoding. Other new work suggests important mechanistic connections to oxidative stress, autoimmunity and cancer, embryonic stem cell pluripotency, and tissue-specific developmental effects. We consider that, for some of its complex functions, variation in RNAP III activity levels lead to nonuniform changes in tRNAs that can shift the translation profiles of key codon-biased mRNAs with resultant phenotypes or disease states.

Distinguishing Core and Holoenzyme Mechanisms of Transcription Termination by RNA Polymerase III

ABSTRACT Transcription termination by RNA polymerase (Pol) III serves multiple purposes; it delimits interference with downstream genes, forms 3′ oligo(U) binding sites for the posttranscriptional processing factor, La protein, and resets the polymerase complex for reinitiation. Although an interplay of several Pol III subunits is known to collectively control these activities, how they affect molecular function of the active center during termination is incompletely understood. We have approached this using immobilized Pol III-nucleic acid scaffolds to examine the two major components of termination, transcription pausing and RNA release. This allowed us to distinguish two mechanisms of termination by isolated Saccharomyces cerevisiae Pol III. A core mechanism can operate in the absence of C53/37 and C11 subunits but requires synthesis of 8 or more 3′ U nucleotides, apparently reflecting inherent sensitivity to an oligo(rU·dA) hybrid that is the termination signal proper. The holoenzyme mechanism requires fewer U nucleotides but uses C53/37 and C11 to slow elongation and prevent terminator arrest. N-terminal truncation of C53 or point mutations that disable the cleavage activity of C11 impair their antiarrest activities. The data are consistent with a model in which C53, C37, and C11 activities are functionally integrated with the active center of Pol III during termination.

Evolving specificity of tRNA 3-methyl-cytidine-32 (m3C32) modification: a subset of tRNAsSer requires N6-isopentenylation of A37

Post-transcriptional modifications of anticodon loop (ACL) nucleotides impact tRNA structure, affinity for the ribosome, and decoding activity, and these activities can be fine-tuned by interactions between nucleobases on either side of the anticodon. A recently discovered ACL modification circuit involving positions 32, 34, and 37 is disrupted by a human disease-associated mutation to the gene encoding a tRNA modification enzyme. We used tRNA-HydroSeq (-HySeq) to examine (3)methyl-cytidine-32 (m(3)C32), which is found in yeast only in the ACLs of tRNAs(Ser) and tRNAs(Thr) In contrast to that reported for Saccharomyces cerevisiae in which all m(3)C32 depends on a single gene, TRM140, the m(3)C32 of tRNAs(Ser) and tRNAs(Thr) of the fission yeast S. pombe, are each dependent on one of two related genes, trm140(+) and trm141(+), homologs of which are found in higher eukaryotes. Interestingly, mammals and other vertebrates contain a third homolog and also contain m(3)C at new sites, positions 32 on tRNAs(Arg) and C47:3 in the variable arm of tRNAs(Ser) More significantly, by examining S. pombe mutants deficient for other modifications, we found that m(3)C32 on the three tRNAs(Ser) that contain anticodon base A36, requires N(6)-isopentenyl modification of A37 (i(6)A37). This new C32-A37 ACL circuitry indicates that i(6)A37 is a pre- or corequisite for m(3)C32 on these tRNAs. Examination of the tRNA database suggests that such circuitry may be more expansive than observed here. The results emphasize two contemporary themes, that tRNA modifications are interconnected, and that some specific modifications on tRNAs of the same anticodon identity are species-specific.

Comment on “Mechanism of eukaryotic RNA polymerase III transcription termination”

Nielsen et al. (Reports, 28 June 2013, p. 1577) characterized their RNA polymerase III (Pol III) preparation and concluded that it requires an RNA hairpin/duplex structure for terminating transcription. We could not corroborate their findings using bona fide Pol III from two laboratory sources. We show that Pol III efficiently terminates transcription in the absence of a hairpin/duplex in vitro and in vivo.

Factors That Shape Eukaryotic tRNAomes: Processing, Modification and Anticodon–Codon Use

Transfer RNAs (tRNAs) contain sequence diversity beyond their anticodons and the large variety of nucleotide modifications found in all kingdoms of life. Some modifications stabilize structure and fit in the ribosome whereas those to the anticodon loop modulate messenger RNA (mRNA) decoding activity more directly. The identities of tRNAs with some universal anticodon loop modifications vary among distant and parallel species, likely to accommodate fine tuning for their translation systems. This plasticity in positions 34 (wobble) and 37 is reflected in codon use bias. Here, we review convergent evidence that suggest that expansion of the eukaryotic tRNAome was supported by its dedicated RNA polymerase III transcription system and coupling to the precursor-tRNA chaperone, La protein. We also review aspects of eukaryotic tRNAome evolution involving G34/A34 anticodon-sparing, relation to A34 modification to inosine, biased codon use and regulatory information in the redundancy (synonymous) component of the genetic code. We then review interdependent anticodon loop modifications involving position 37 in eukaryotes. This includes the eukaryote-specific tRNA modification, 3-methylcytidine-32 (m3C32) and the responsible gene, TRM140 and homologs which were duplicated and subspecialized for isoacceptor-specific substrates and dependence on i6A37 or t6A37. The genetics of tRNA function is relevant to health directly and as disease modifiers.