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RNA Metabolism in Cell Biology, Growth, and Development

• Richard J. Maraia, MD, Head, Section on Molecular and Cellular Biology
• Sergei Gaidamakov, PhD, Biologist
• Sandy Mattijssen, PhD, Research Fellow
• Nathan Blewett, PhD, Postdoctoral Fellow
• Abdul Khalique, PhD, Visiting Fellow
• Saurabh Mishra, PhD, Visiting Fellow
• Amitabh Ranjan, PhD, Visiting Fellow
• Rima Sakhawala, BS, Postbaccalaureate Fellow
• Carissa Stover, BS, Postbaccalaureate Fellow

We are interested in tRNAs and certain mRNAs as well as some of their key interacting proteins and how the pathways involved in their biogenesis, maturation, and metabolism intersect with processes critical to cell proliferation, growth, and development during health and disease. One focus is the synthesis of tRNAs by RNA polymerase III (RNAPIII), as well as the early phases of their post-transcriptional processing and 'handling' by the eukaryote-ubiquitous RNA–binding protein known as La. The La protein was first discovered because, in some individuals, it becomes a target of autoantibodies and is part of an autoimmune process that leads to (and is diagnostic of) Sjögren’s syndrome, systemic lupus erythematosus (SLE), and neonatal lupus. The autoimmunity to La occurs by an as yet undetermined mechanism, and the La protein is sometimes referred to as the La autoantigen. Beyond this, and critical to its normal essential function in vertebrates and other eukaryotes, La contains RNA–binding motifs as well as subcellular trafficking elements. La associates with noncoding (nc) RNA as well as with mRNAs, presumably to coordinate activities in the nucleus and cytoplasm. In the nucleus, La binds to the 3′ oligo(U) motif common to all RNAP III transcripts and functions by protecting its RNA ligands, principally the nascent precursor tRNAs, from 3′ exonucleolytic decay and by serving as a chaperone to prevent their misfolding. The major products of RNAP III are the tRNAs, although it also synthesizes 5S rRNA and certain other ncRNAs. We also investigate the biochemistry, genetics, and function of specific post-transcriptional modifications that impact tRNA function during translation of mRNAs into protein by the ribosome. We also study La–related protein-4 (LARP4), which is predominantly cytoplasmic, interacts with the 3′ poly(A) tails of mRNAs, and contributes to their stability/metabolism and translational control [Reference 1].

In summary, we strive to understand the structure-function relationship, genetics, cell- and molecular biology of the La protein, LARP4, the tRNA modification enzymes tRNA isopentenyltransferase TRIT1, the tRNA–modification enzyme Trm1, and their contributions to growth and development. We use genetics, cell and structural biology, and biochemistry in model systems that include yeast, human and mouse tissue culture cells, and gene-altered mice.

Data from our lab suggest that the levels of cytoplasmic tRNA may uniquely regulate the translation of LARP4 mRNA (and other mRNAs), which in turn promotes stabilization of mRNAs encoding ribosomal proteins [Reference 1]. Tumor suppressors and oncogenes mediate deregulation of tRNA production by RNAP III, collectively contributing to the increased translational capacity required for proliferation during growth and development and of cancer cells. However, tRNA levels/abundance alone do not account for their differential nor their regulated activity. tRNAs are the most heavily and the most diversely modified molecules in cells, and some of the modifications can control or regulate their codon-specific activity. As alluded to above, we study some of the key modifications that affect tRNA translational activity during mRNA decoding.

The tRNA–modification enzyme Trm1 synthesizes dimethyl-guanosine-26 (m2,2G26), which resides at the top of the anticodon stem of several tRNAs, and TRIT1 adds an isopentenyl group onto Adenine-37 in the anticodon loop of certain tRNA (i6A37) molecules. We showed that both modifications can activate their tRNAs for translation in codon-specific assays. We also examine effects of TRIT1 on tRNA activity during mammalian development, using trit1-gene–altered mice as a model system in which to understand how a deficiency in the enzyme leads to childhood mitochondrial dysfunction and metabolic disease. We complement our studies by investigating how differences in the copy numbers of tRNA genes can affect how the genetic code is deciphered via use of secondary information in the genetic code. In humans they number more than 500, and are distributed on all the chromosomes, many residing in clusters, the loci of which can vary, as do the loci of individual tRNA gene loci.

A model emerges from the integration of our studies whereby differential expression of tRNAs in a tissue- and temporal-specific manner, together with their differential modifications, controls mRNA decoding in a codon enrichment–specific hierarchical manner, which determines the translational output of mRNA transcriptomes of the corresponding cell types and tissues during growth and development, as well as in health and disease states. Shifts or perturbations in the levels of specific tRNAs or subsets/clusters thereof and/or of their modifications can affect shifts in the hierarchical translation of mRNAs. In terms of genetics, perturbations in a tRNA's expression and/or activity can be considered a potential modifier of a conventional disease-associated allele. Therefore, a vision for the future of genetics and medicine is that variances among the tRNAomes of individuals should be considered as sources contributing to disease penetrance, especially to multifactorial and multigene disorders.

Activities of RNA polymerase III (RNAP III) and associated factors

The RNAP III enzyme consists of 17 subunits, several with homology to subunits of RNAPs I and II. In addition, the transcription factor TFIIIC, composed of six subunits, binds to A- and B-box promoters (promoter elements of tRNA genes) and recruits TFIIIB to direct RNAP III to the correct start site. TFIIIB–RNAP III complexes appear highly stable and demonstrate great productivity in supporting the many cycles of initiation, termination, and re-initiation necessary to produce the more than ten-fold molar excess of tRNAs relative to ribosomes that is required to drive translation during growth and development. In contrast to all other multisubunit RNA polymerases, termination and reinitiation by RNAP III (also known as Pol III) are functionally if not physically linked. Our laboratory has developed methods for in vivo and biochemical studies to examine the unique mechanisms used by RNAP III. Hereditary mutations in RNAP III cause hypomyelinating leukodysplasia, as well as defects in innate immunity. In addition to its essentiality for cell proliferation, RNAP III is also linked to aging.

Transcription termination delineates 3′ ends of gene transcripts, prevents otherwise runaway RNAP from intruding into downstream genes and regulatory elements, and enables release of the RNAP for recycling. While other RNAPs require complex cis signals and/or accessory factors to accomplish these activities, eukaryotic RNAP III does so autonomously with high efficiency and precision at a simple oligo(dT) stretch of 5–6 bp. A basis for this high-density cis information is that both the template and non-template strands of the RNAP III terminator carry distinct signals for different stages of termination. High-density cis information is a feature of the RNAP III system that is also reflected by dual functionalities of the tRNA promoters as both DNA and RNA elements. Furthermore, the TFIIF–like RNAP III subunit C37 is required for this function of the non-template strand signal. Our results reveal the RNAP III terminator to be 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.

Control of the differential abundance or activity of tRNAs can be an important determinant of gene regulation. RNAP III synthesizes all tRNAs in eukaryotes, and its derepression is associated with cancer. Maf1 is a conserved general repressor of RNAP III under the control of TOR (target of rapamycin), which acts to integrate transcriptional output and protein-synthetic demand toward metabolic economy. We used tRNA-HydroSeq to document that little change occurred in the relative levels of different tRNAs in maf1Δ cells. By contrast, the efficiency of N2,N2-dimethyl G26 [m(2)2G26)] modification on certain tRNAs was reduced in response to maf1 deletion and associated with anti-suppression, which we validated by other methods. Over-expression of Trm1, which produces m(2)2G26, reversed maf1 anti-suppression. The model that emerges is that competition by elevated tRNA levels in maf1Δ cells leads to m(2)2G26 hypo-modification resulting from limiting Trm1, thus reducing the activity of suppressor tRNASerUCA (UCA is the anticodon for serine) and accounting for anti-suppression. Consistent with this, RNAP III mutations associated with hypo-myelinating leukodystrophy reduce tRNA transcription, increase m(2)2G26 efficiency, and reverse anti-suppression. Extending this more broadly, a reduction in tRNA synthesis by treatment with rapamycin leads to increased m(2)2G26 modification, and this response is conserved among highly divergent yeasts and human cells [Reference 6].

The ability of RNAP III to efficiently recycle from termination to reinitiation is critical for abundant tRNA production during cellular proliferation, development, and cancer. We used two tRNA–mediated suppression systems to screen for Rpc1 (subunit of RNAP III) mutants with gain- and loss- of termination phenotypes in Schizosaccharomyces pombe. We mapped 122 point mutation mutants to a recently solved 3.9 Å structure of the yeast RNAP III elongation complex (EC); they cluster in the active center bridge helix and trigger loop, as well as in the pore and funnel formation, the latter indicating involvement in termination of the RNA–cleavage domain of the C11 subunit. Biochemical kinetic and genetic data indicate that mutants with the RT (readthrough) phenotype synthesize more RNA than wild-type cells and, surprisingly, more than can be accounted for by the mutants' increased elongation rate. Importantly, similar mutations in spontaneous cancer suggest this as an unforeseen mechanism of RNAP III activation in disease.

The role of La-related protein-4 (LARP4) in poly(A)-mediated mRNA stabilization

Ubiquitous in eukaryotes, La proteins are involved in two broad functions: (1) metabolism of a wide variety of precursor tRNAs and other small nuclear RNAs by association with these RNAs’ common UUU-3′ OH–transcription termination elements; and (2) translation of specific subsets of mRNAs, such as those containing 5′ IRES (internal ribosome entry site) motifs. La-related protein-4 (LARP4) emerged later in evolution, and we found it to be an mRNA–associated cytoplasmic factor associated with poly(A)–binding protein C1 (PABPC1). LARP4 uses two regions to bind to PABPC1. We showed that the N-terminal domain of LARP4, comprising amino acids 1–286 and containing two RNA–binding motifs known as an 'La module,' exhibits preferential binding to poly(A). The La module is flanked on each side by a different motif that independently interacts with PABP. LARP4 is controlled at the level of mRNA stability: one level of control is by an A+U-rich element (ARE) in its 3′ UTR via interactions with the protein tristetraproline (TTP), which is regulated in mammalian cells through TTP by tumor necrosis factor alpha (TNFa); a second level of control was found for LARP4 mRNA coding sequence in an unusual group of synonymous codons with poor match to cellular tRNA levels [Reference 1]. The LARP4 protein controls the metabolism/homeostasis and translation of heterologous mRNAs by affecting their poly(A) tail length [Reference 1].

Fission yeast as a model system for the study of tRNA metabolism and function in translation

More than 20 years ago, we began developing, refining, and advancing a tRNA–mediated suppression (TMS) system in fission yeast (Schizosaccharomyces pombe), which provides a red-white phenotypic real-time assay that can be used to investigate various aspects of tRNA biogenesis, maturation, and metabolism of tRNAs in vivo. In fission yeast, the human La protein can replace the tRNA processing/maturation function of Sla1p, the fission yeast equivalent of the La protein. Moreover, in fission yeast, human La is faithfully phosphorylated on Ser-366 by protein kinase CKII, the same enzyme that phosphorylates Ser-366 in human cells, and this phosphorylation event promotes pre-tRNA processing. We use this system to study transcription by RNAP III, post-transcriptional processing, and tRNA modifications by conserved enzymes that produce tRNA isopentenyl-adenosine-37 and dimethyl-guanosine-26.

tRNAs, codon use, and mRNA metabolism in growth and development

A major interest is in deciphering what we refer to as 'secondary information' in the genetic code, information that is derived from mRNAs' biased use of synonymous codons. This can produce a layer of information beyond the amino acid sequence of a protein; i.e., in addition to providing the template for the sequence of a protein, the use of certain synonymous codons can also produce additional biochemical effects, which we refer to as 'secondary information.' The effects can be related to ribosome pausing, which can affect protein folding, or to alterations in the stability of the mRNA. Other types of secondary information can also be encoded in synonymous codons; for example, sets of mRNAs that share similar patterns of synonymous codon bias are similarly sensitive to tRNAs with the same anticodon modification and exhibit similar patterns of efficiency of translation elongation. The components of the secondary information system are the tRNA pool, the tRNA–modification enzymes, and the codon bias distribution among the mRNAs. We recently found that synonymous codon use by the human LARP4 mRNA is a key determinant in the control of the expression levels of its mRNA and protein, and that increases in otherwise limiting tRNAs that are cognate to these codons increases LARP4 production. This in turn activates LARP4 to promote a net increase in the poly(A) tail length of heterologous mRNAs, including those that encode ribosomal protein subunits [Reference 1]. This may be important because ribosome production is regulated during growth and development, and the potential circuit involving LARP4 control by tRNA could be an important point of control.

Publications

1. Mattijssen S, Arimbasseri AG, Iben JR, Gaidamakov S, Lee J, Hafner M, Maraia RJ. LARP4 mRNA codon-tRNA match contributes to LARP4 activity for ribosomal protein mRNA poly(A) tail length protection. eLIFE 2017;6:e28889.
2. Blewett NH, Iben JR, Gaidamakov S, Maraia RJ. La deletion from mouse brain alters pre-tRNA metabolism and accumulation of pre-5.8S rRNA, with neuron death and reactive astrocytosis. Mol Cell Biol 2017;37:e00588.
3. Rijal K, Maraia RJ. Active center control of termination by RNA polymerase III and tRNA gene transcription levels in vivo. PLoS Genet 2016;12:e1006253.
4. Lamichhane TN, Arimbasseri AG, Rijal K, Iben JR, Wei FY, Tomizawa K, Maraia RJ. Lack of tRNA-i6A modification causes mitochondrial-like metabolic deficiency in S. pombe by limiting activity of cytosolic tRNATyr, not mito-tRNA. RNA 2016;2:583-596.
5. Arimbasseri AG, Maraia RJ. Mechanism of transcription termination by RNA polymerase III utilizes a non-template strand sequence-specific signal element. Mol Cell 2015;58:1124-1132.
6. Arimbasseri AG, Blewett NH, Iben JR, Lamichhane TN, Cherkasova V, Hafner M, Maraia RJ. RNA polymerase III output is functionally linked to tRNA dimethyl-G26 modification. PLoS Genetics 2015;11:e1005671.

Collaborators

• Maria R. Conte, PhD, King's College, University of London, London, United Kingdom
• Markus Hafner, PhD, Laboratory of Muscle Stem Cells and Gene Regulation, NIAMS, Bethesda, MD
• James R. Iben, PhD, Molecular Genomics Core, NICHD, Bethesda, MD
• Robert Tylor, PhD, Newcastle University, Newcastle, United Kingdom

Contact

For more information, email maraiar@mail.nih.gov or visit http://maraialab.nichd.nih.gov.

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