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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

We are interested in the biogenesis and metabolism pathways for tRNAs and certain mRNAs and how these intersect with pathways related to cell proliferation, growth, and development. We focus on the synthesis of tRNAs by RNA polymerase (RNAP) III, as well as the early phases of their post-transcriptional handling by the RNA–binding protein La, and certain modifications that impact their function in the translation of mRNAs into protein by the ribosome. The La protein has been conserved throughout eukaryotic evolution, from single-cell intracellular parasites, yeast, ancient animal cells, plants, and all higher eukaryotes. In some people, the La protein becomes a target of autoantibodies, such as in (and is diagnostic of) patients with Sjögren’s syndrome, systemic lupus erythematosus (SLE), and neonatal lupus. Critical to its normal function, the La protein contains several nucleic acid–binding motifs as well as numerous subcellular trafficking elements and it associates with noncoding (nc) RNA as well as with mRNAs 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. In addition to the major products of RNAP III, which are the tRNAs, it also synthesizes 5S rRNA and certain other ncRNAs. We also study La–related protein-4 (LARP4), which is predominantly cytoplasmic and interacts with the 3′ poly(A) region of certain mRNAs and contributes their 3′ end metabolism and translational control (Reference 1).

We strive to understand the structure-function relationship and the cell and molecular biology of the La protein, LARP4, the tRNA isopentenyltransferase TRIT1, and 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 tissue culture cells, and gene-altered mice.

Recent data from our lab suggest that the levels of cytoplasmic tRNA may uniquely regulate the translation of LARP4 mRNA, which in turn promotes stabilization of mRNAs encoding ribosomal proteins (Reference 1). Tumor suppressors and oncogenes mediate deregulation of transcript production by RNAP III, the RNAP responsible for tRNA synthesis, thus contributing to increased capacity for proliferation of cancer cells. However, tRNA levels alone do not account for their differential nor regulated activity. tRNAs are the most heavily and the most diversely modified molecules in cells. We also study some of the key modifications that affect their translational activity.

We also focus on other aspects of tRNA biogenesis and function, such as Trm1, which synthesizes dimethyl-guanosine-26 (m2,2G26) in several tRNAs, and TRIT1, which adds an isopentenyl group onto Adenine-37 in the anticodon loop of certain tRNA (i6A37) molecules. We are examining the effects of TRIT1 on tRNA activity in translation in a codon-specific manner, as well as during mammalian development, using trit1-gene-altered mice as a model system, to try to understand how its deficiency leads to childhood mitochondrial dysfunction and metabolic disease. The studies are complemented by investigating how differences in the copy numbers of tRNA genes, which in humans is greater than 600 interspersed on all of the chromosomes of which many reside in clusters and which indeed vary (at the cluster and individual tRNA gene loci) among humans, can affect how the genetic code is deciphered via use of secondary information in the genetic code.

A model that emerges from 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 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 as 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 potential sources contributing to disease penetrance, especially to multifactoral and multigene disorders.

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

The RNAP III enzyme consists of 17 subunits, several with strong homology to subunits of RNAPs I and II. In addition, the transcription factor TFIIIC, composed of six subunits, binds to the A- and B-box promoters and recruits TFIIIB to direct RNAP III to the correct start site. RNAP III complexes are highly stable and demonstrate great productivity in supporting many cycles of initiation, termination, and re-initiation. For example, each of the 5S rRNA genes in human cells must produce approximately 10⁴ to 10⁵ transcripts per cell division to provide sufficient 5S rRNA for ribosomes. While RNAPs I, II, and III are homologous, their properties are distinct in accordance with the unique functions related to the different types of gene they transcribe. Given that some mRNA genes can be hundreds of kilobase-pairs long, RNAP II must be highly processive and avoid premature termination. RNAP II terminates in response to complex termination/RNA–processing signals that require endo-nucleolytic cleavage of RNA upstream of the elongating polymerase. By contrast, formation of the UUU–3′OH terminus of nascent RNAP III transcripts appears to occur at the RNAP III active center. The dT(n) tracts at the ends of class III genes directly signal pausing and release by RNAP III such that termination and RNA 3′-end formation are coincident and efficient.

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. 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.

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 was 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 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. We published this work in 2015.

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 S. 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 their increased elongation rate. Importantly, similar mutations in spontaneous cancer suggest this as an unforeseen mechanism of RNAP III activation in disease.

La-related protein-4 (LARP4) in translation-coupled mRNA stabilization

Ubiquitous in eukaryotes, the 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 motifs. LARP4 emerged later in eukaryotic evolution and is conserved in vertebrates as an mRNA–associated cytoplasmic translation factor. We showed that the two RNA–binding motifs of LARP4, which work together as a ‘La module,’ exhibit preferential binding to poly(A) and are flanked on each side by a different motif that independently interacts with the poly(A)–binding protein (PABP). LARP4 is controlled at the level of mRNA stability by the protein tristetraproline (TTP) and is regulated in mammalian cells through TTP by the tumor necrosis factor alpha (TNFa). LARP4 controls mRNA metabolism/homeostasis and translation.

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

About 22 ago, we began developing and have since been refining and advancing a tRNA–mediated suppression system in fission yeast (Schizosaccharomyces pombe) that provides a red-white phenotypic real-time assay in vivo. In fission yeast, the human La protein can replace the tRNA processing/maturation function of Sla1p, the fission yeast 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 the phosphorylation promotes pre-tRNA processing. We use this system to study other aspects of tRNA biogenesis, including transcription by RNAP III, post-transcriptional processing, and tRNA modifications by the conserved enzymes that produce tRNA isopentenyl-adenosine-37 and dimethyl-guanosine-26. The antitumor drug rapamycin inhibits the master growth regulator and signal integrator TOR, which coordinates ribosome biogenesis and protein-synthetic capacity with nutrient homeostasis and cell-cycle progression. Rapamycin inhibits proliferation of the yeast Saccharomyces cerevisiae and of human cells, whereas proliferation of the yeast S. pombe is resistant to rapamycin. We found that deletion of the tit1 gene, which encodes tRNA isopentenyltransferase, causes S. pombe proliferation to become sensitive to rapamycin, with a ‘wee’ phenotype (smaller than normal cells as a result of premature entry into mitosis), suggesting a cell-cycle defect. The gene product of tit1 is a homolog of S. cerevisiae MOD5 (a tRNA dimethylallyltransferase), the human tumor suppressor TRIT1, and the Caenorhabditis elegans life-span gene product GRO-1, enzymes that isopentenylate N6-adenine-37 (i6A37) in the anticodon loop of a small subset of tRNAs. Anticodon loop modifications are known to affect codon-specific decoding activity. Indicating a requirement for i6A37 for optimal codon-specific translation efficiency, as well as defects in carbon metabolism related to respiration, tit1^Δ cells exhibit anti-suppression. Genome-wide analyses of gene-specific enrichment of codons cognate to i6A37–modified tRNAs identify genes involved in ribosome biogenesis, carbon/energy metabolism, and cell-cycle genes, congruous with tit1^Δ phenotypes. We found that mRNAs enriched in codons cognate to i6A37–modified tRNAs are translated less efficiently than mRNAs with low content of the cognate codons. We determined that the Tit1p–modified tRNA Tyr exhibits about five-fold higher specific decoding activity during translation than the unmodified tRNA Tyr.

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. This information derives from mRNAs' choice of synonymous codons that encode the same amino acid. This can produce a layer of information beyond providing 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 alterations in the stability of the mRNA. Other types of secondary information can also be encoded in the use of 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 of 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 relevant 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. Maraia RJ, Mattijssen S, Cruz-Gallardo I, Conte MR. The La and related RNA-binding proteins (LARPs): structures, functions, and evolving perspectives. Wiley Interdiscip Rev RNA 2017 8(6):e1430.
3. 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:e00588-16.
4. 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.
5. Arimbasseri AG, Iben J, Wei FY, Rijal K, Tomizawa K, Hafner M, Maraia RJ. Evolving specificity of tRNA 3-methyl-cytidine-32 (m3C32) modification: a subset of tRNAsSer requires N6-isopentenylation of A37. RNA 2016 22:1400-1410.
6. Maraia RJ, Rijal K. Structural biology: a transcriptional specialist resolved. Nature 2015 528:204-205.
7. 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.

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
• Brant Weinstein, PhD, Section on Vertebrate Organogenesis, NICHD, Bethesda, MD

Contact

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

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