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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, Staff Scientist
• Abdul Khalique, PhD, Visiting Fellow
• Saurabh Mishra, PhD, Visiting Fellow
• Amitabh Ranjan, PhD, Visiting Fellow
• Alan Kessler, PhD, Fellow
• Jack Prochnau, BS, Postbaccalaureate Fellow

We are interested in tRNAs and mRNAs as well as some of their 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 (RNAP III), as well as their post-transcriptional processing and ‘handling’ by the eukaryotic RNA–binding protein La. The protein was discovered because it is a target of autoantibodies in patients who suffer from, is integral to an autoimmune process that leads to, and is diagnostic of Sjögren’s syndrome, systemic lupus erythematosus (SLE), and neonatal lupus. Autoimmunity to La occurs by a complex, as yet incompletely understood mechanism, and the protein is sometimes referred to as the La autoantigen. Critical to its normal essential function, the conserved La protein contains multiple RNA–binding motifs and subcellular trafficking elements, and associates with noncoding (nc) RNAs, mostly in the nucleus, as well as with mRNAs in the cytoplasm. In the nucleus, La binds to the 3′ oligo(U) motif common to all RNAP III transcripts as discrete small RNPs (ribonucleoproteins) and functions by protecting its most abundant ligands, the nascent precursor tRNAs, from 3′ exonucleolytic digestion and by serving as a chaperone to prevent their misfolding. Although the major products of RNAP III are the tRNAs, it also synthesizes 5S rRNA and some other essential noncoding RNAs (ncRNAs) involved in fundamental processes necessary for translating the genetic information in mRNA during protein synthesis.

Our investigations also include specific post-transcriptional modifications of tRNAs that impact their metabolism and function during translation by cytoplasmic and mitochondrial ribosomes. We also study La–related protein-4 (LARP4), which, in contrast to La protein, is predominantly cytoplasmic at steady state, and interacts mostly with mRNAs rather than ncRNAs. However, similar to La, LARP4 interacts with the 3′ end regions of its RNA ligands, in this case the mRNA poly(A) tails (PATs), and contributes to their stability/metabolism and translation. Genome-wide mRNA-Seq and analysis of PATs indicate that LARP4 interacts with a large number of mRNAs and promotes their stability. Such analyses of mice whose LARP4 gene is disrupted reveal shorter PATs, whereas over-expression of LARP4 leads to PAT lengthening. Studies on the mechanisms by which mRNA PAT metabolism and mRNA stability are linked suggest that LARP4 affects nascent PAT metabolism. Mechanistic studies are under way.

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 and 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 levels of cytoplasmic tRNAs may regulate translation-mediated decay of LARP4 mRNA and LARP4 levels, which in turn promotes stabilization of mRNAs encoding ribosomal proteins. 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 or their 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 translation 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 (m^2,2G26), which resides at the top of the anticodon stem of several tRNAs, whereas TRIT1 adds an isopentenyl group onto adenine-37 in the anticodon loop of certain tRNA (i⁶A37) molecules. We showed that each of these modifications can activate their tRNAs for translation in codon-specific assays. We also examine effects of TRIT1 on tRNA activity during mammalian development, using TRIT-gene–altered mice as a model system to understand how a deficiency in this 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, active tRNA genes number more than 300, and are distributed on all chromosomes, many residing in clusters, the loci of which can vary, as do the loci of individual tRNA genes. A system we use to examine codon-specific effects of loss of the i⁶A37 modification on a specific tRNA is the fission yeast Schizosaccharomyces pombe, in which human TRIT1 can complement the phenotype caused by deletion of the homologous enzyme Tit1. We also use the S. pombe system to study La and pol III, in which the human La protein can complement the phenotype caused by deletion of the fission-yeast La-homolog Sla1.

One theme of our work and that of others is that differential expression of tRNAs occurs in a tissue- and temporal-specific manner and, 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. 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 tRNA gene copy numbers of individuals should be considered as contributing to disease penetrance, especially to multifactorial and multigene disorders.

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

The RNAP III multisubunit enzyme complex consists of 17 subunits, several with homology to subunits of RNAPs I and II. 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 tenfold 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 re-initiation 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 being essential 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 in the 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 (hydrolysis-based tRNA sequencing) to document that little change occurred in the relative levels of different tRNAs in maf1 mutated 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. Overexpression of Trm1, which produces m^2,2G26, reversed maf1 anti-suppression. The model that emerges is that competition by elevated tRNA levels in maf1-delta cells leads to m^2,2G26 hypomodification 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 hypomyelinating 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, a response that is conserved among highly divergent yeasts and human cells [Reference 6].

The ability of RNAP III to efficiently recycle from termination to re-initiation 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 of RNAP III. 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: first, 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 second, translation of specific subsets of mRNAs, such as those containing 5′ IRES (internal ribosome entry site) motifs. LARP4 emerged later in evolution, and we found it to be an mRNA–associated cytoplasmic factor associated with poly(A)–binding protein C1 (PABPC1, PABP). LARP4 uses two regions to bind to PABPC1. We showed that the N-terminal domain (NTD, amino acids 1-286) of LARP4, consisting of an N-terminal region (NTR, amino acids 1-111) followed by two tandem RNA–binding motifs known as an 'La module' (111–285), exhibits preferential binding to poly(A). The NTR contains a unique PAM2w motif that binds to the MLLE (a peptide-binding domain) of PABP. The group of our collaborator Maria Conte showed that the N-terminal region (NTR) itself is responsible for most of the poly(A) binding and that, moreover, this involves conserved residues unique to the PAM2w of LARP4. The La module is flanked by a different motif on each side that independently interact 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), the latter of which is regulated in mammals by tumor necrosis factor alpha (TNFα); a second level of control was found for the 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. Working with researchers in the NICHD Molecular Genomics Core facility, we developed a single-molecule, high-throughput nucleotide-resolution poly(A)-tail sequencing method referred to as SM-PAT-Seq, which yielded insights into LARP4 function and mechanism. LARP4 is a global factor involved in mRNA poly(A) length homeostasis and appears to effect mRNA stabilization by opposing the action of deadenylases when poly(A) tails are short.

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 S. 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 S. pombe, the human La protein can replace the tRNA–processing/maturation function of Sla1p, the S. pombe equivalent of the La protein. Moreover, in S. pombe, 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 the 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 of ours is to decipher 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 increase 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], which 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, Iben JR, Li T, Coon SL, Maraia RJ. Single molecule poly(A) tail-seq shows LARP4 opposes deadenylation throughout mRNA lifespan with most impact on short tails. eLife 2020;9:e59186.
2. Khalique A, Mattijssen S, Haddad AF, Chaudhry S, Maraia RJ. Targeting mitochondrial and cytosolic substrates of TRIT1 isopentenyltransferase: specificity determinants and tRNA-i6A37 profiles. PLoS Genet 2020;16:e1008330.
3. Mishra S, Maraia RJ. RNA polymerase III subunits C37/53 modulate rU:dA hybrid 3' end dynamics during transcription termination. Nucleic Acids Res 2019;47:310–327.
4. 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.
5. Mattijssen S, Kozlov G, Gaidamakov S, Ranjan A, Fonseca BD, Gehring K, Maraia RJ. The isolated La-module of LARP1 mediates 3’ poly(A) protection and mRNA stabilization, dependent on its intrinsic PAM2 binding to PABPC1. RNA Biol 2020;in press.
6. Blewett NH, Maraia RJ. La involvement in tRNA and other RNA processing events including differences among yeast and other eukaryotes. Biochim Biophys Acta Gene Regul Mech 2018;1861:361–372.

Collaborators

• Maria R. Conte, PhD, King's College, University of London, London, United Kingdom
• Steven Coon, PhD, Molecular Genomics Core, NICHD, Bethesda, MD
• James R. Iben, PhD, Molecular Genomics Core, NICHD, Bethesda, MD

Contact

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

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