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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
• Saurabh Mishra, PhD, Visiting Fellow
• Keshab Rijal, PhD, Visiting Fellow
• Alex Haddad, 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 the RNA polymerase (RNAP) III, the early phases of their post-transcriptional handling by the RNA–binding protein La, and certain modifications that impact their translational function. The La protein is highly conserved in eukaryotic evolution and is a target of autoantibodies in (and diagnostic of) patients with Sjögren’s syndrome, systemic lupus erythematosus (SLE), and neonatal lupus. It contains several nucleic acid–binding motifs as well as numerous subcellular trafficking elements and it associates with noncoding (nc) and messenger RNAs to coordinate activities in the nucleus and cytoplasm. La protein binds to the 3′ oligo(U) motif common to all RNAP III transcripts and functions by protecting its RNA ligands, principally the large variety of nascent precursor tRNAs, from 3′-exonucleolytic decay and by serving as a chaperone to prevent their misfolding. In addition to its major products, the tRNAs and 5S rRNA, RNAP III also synthesizes certain other non-coding RNAs. La-related protein-4 (LARP4) interacts with the 3′ poly(A) region of certain mRNAs and contributes their 3′ end metabolism and translational control (Yang R et al. Mol Cell Biol 2011;31:542).

We focus on the tRNA–modification enzymes Trm1, which synthesizes dimethyl-guanosine-26 (m2,2G26) in several tRNAs, and tRNA isopentenyltransferase (TRIT1), which modifies tRNA by adding an isopentenyl group onto adenine at position 37 of certain tRNA (i6A37) molecules. We are examining the effects of TRIT1 on tRNA activity in translation in a codon-specific manner (Lamichhane TN et al. Mol Cell Biol 2013;33:4900; Lamichhane TN et al. Mol Cell Biol 2013;33:2918), as well as during mammalian development, and how its deficiency leads to childhood mitochondrial dysfunction and metabolic disease. We are also investigating how differences in the copy number of tRNA genes, which in humans is greater than 500 interspersed on all of the chromosomes and which indeed varies among humans (Iben JR, Maraia RJ. Gene 2014;536:376), can affect how the genetic code is deciphered. 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.

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

Functions of the La antigen in RNA expression and mammalian cell viability

Previous findings regarding nucleolar localization, cytoplasmic splicing, and retrograde transport indicated that the tRNA production pathway is more complex in its biochemistry, spatial organization, and sequential order than previously thought. By binding to UUU–3′ OH, the La protein shields newly transcribed pre–tRNAs from 3′-end digestion and functions as a chaperone for misfolded or otherwise imperfect pre–tRNAs. It has thus become clear that La serves the tRNA pathway at several levels, including protection of pre–tRNAs from 3′ exonucleases; nuclear retention of pre–tRNAs, thereby preventing premature export of pre–tRNAs; and promotion of a newly identified processing step distinct from 3′-end protection.

Studies in gene-altered mice revealed that La is required for cell survival in developing B cells of the immune system and in post-mitotic cells in the cerebral cortex in the developing brain (Gaidamakov S et al. Mol Cell Biol 2014;34:123). Our more recent data indicate that severe loss of frontal cortex brain mass in conditional La–deleted mice is associated with perturbations in rRNA processing, and is followed by robust innate immune reactivity, including astrocyte invasion.

To study RNAP III– and La–dependent tRNA biogenesis, we developed a red-white tRNA–sensitive reporter system in the fission yeast Schizosaccharomyces pombe (Figure 1), a yeast that generally appears more similar to the human organism than does another yeast, Saccharomyces cerevisiae, with respect to cell-cycle control, gene-promoter structure, and the complexity of pre–mRNA splicing. From sequence analysis of RNAP III–transcribed genes, we predicted and then confirmed that RNAP III termination–signal recognition in S. pombe would be more similar to human RNAP III than it is for S. cerevisiae RNAP III. Our system is based on tRNA–mediated suppression of a nonsense codon in the suppressor-sensitive nonsense allele ade6-704 and affords the benefits of fission yeast biology while lending itself to certain aspects of 'humanization.' We have been able to study the tRNA processing–associated function of the human La protein (hLa) because it is so highly conserved that it can replace the processing function of the S. pombe La protein Sla1p in vivo.

Briefly, we found that: (1) the human pattern of phosphorylation of hLa at the serine-366 target site by the protein kinase CK2 occurs faithfully in S. pombe and promotes tRNA production; (2) various conserved subcellular trafficking signals in La proteins can be positive or negative determinants of tRNA processing; (3) La can protect pre–tRNAs from the nuclear surveillance 3′ exonuclease Rrp6p; (4) the 3′ exonuclease that processes pre–tRNAs in the absence of fission yeast La protein Sla1p is distinct from Rrp6p; (5) Sla1p is limiting in S. pombe cells, and the extent to which it influences the use of alternative tRNA maturation pathways is balanced by the RNA 3′–5′ cleavage activity of the RNAP III termination–associated RNAP III subunit Rpc11p; and (6) La proteins use distinct RNA–binding surfaces, one on the La motif (LM) and the other on the RNA recognition motif-1 (RRM1), to promote different steps in tRNA maturation.

Results of our previous work suggested that La can use several surfaces, perhaps combinatorially, to engage various classes of RNAs, e.g., pre–tRNAs versus mRNAs, or to perform different functions (Huang Y et al. Nat Struct Mol Biol 2006;13:611; Maraia RJ, Bayfield MA. Mol Cell 2006;21:149). Consistent with this notion, some pre–tRNAs require only the UUU–3′OH binding activity while others depend on a second activity in addition to 3′-end protection that requires an intact RRM (RNA recognition motif) surface to promote a previously unknown step in tRNA maturation. One of our objectives is to identify cellular genes, other than that encoding La, that contribute to this 'second' activity. Toward this goal, we isolated and have begun to characterize S. pombe revertant mutants that overcome a defect in the second activity.

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 (Arimbasseri AG, Maraia RJ. Mol Cell Biol 2013;33:1571).

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 the target of rapamycin (TOR), 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 decreased 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. A model that emerges is that competition by elevated tRNA levels in maf1Δ cells leads to m(2)2G26 hypomodification resulting from limiting Trm1, thus reducing the activity of suppressor-tRNASerUCA 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 and this response is conserved among highly divergent yeasts and human cells (Arimbasseri AG et al. PLoS Genetics 2015;11:e1005671).

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 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, 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 (Reference 1).

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 a 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) (Yang R et al. Mol Cell Biol 2011;31:542). 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 (TNFα) (Mattijssen S, Maraia RF. Mol Cell Biol 2015;36:574). 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 that provides a red-white phenotypic real-time assay in vivo (reviewed in Rijal K et al. Gene 2015;556:35). In fission yeast, the human La protein can replace the tRNA processing/maturation function of the fission yeast La protein Sla1p. 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 (Intine RV et al. Mol Cell 2000;6:339). 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 S. cerevisiae and 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, 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.

Publications

1. 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.
2. 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;22:583-596.
3. 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.
4. Arimbasseri AG, Maraia RJ. RNA polymerase III advances: structural and tRNA functional views. Trends Biochem Sci 2016;41:546-559.
5. Arimbasseri AG, Maraia RJ. A high density of cis-information terminates RNA polymerase III on a 2-rail track. RNA Biol 2016;13:166-171.
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, UK
• 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, UK

Contact

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

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