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RNA Metabolism in Cell Biology, Growth, and Development

• Richard J. Maraia, MD, Head, Section on Molecular and Cellular Biology
• Vera Cherkasova, PhD, Staff Scientist
• Sergei Gaidamakov, PhD, Biologist
• Nathan Blewett, PhD, Postdoctoral Fellow
• Aneeshkumar Arimbasseri, PhD, Visiting Fellow
• Sandy Mattijssen, PhD, Visiting Fellow
• Saurabh Mishra, PhD, Visiting Fellow
• Keshab Rijal, PhD, Visiting Fellow

We are interested in how the biogenesis of and metabolism pathways for RNAs, especially tRNAs and certain mRNAs, intersect with pathways related to cell proliferation, growth, and development. We focus on the synthesis of tRNAs by RNA polymerase (RNAP, Pol) 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 a target of autoantibodies prevalent in (and diagnostic of) patients with Sjögren’s syndrome, systemic lupus, and neonatal lupus. La contains several nucleic acid–binding motifs as well as several subcellular trafficking signals and associates with non-coding and messenger RNAs to coordinate activities in the nucleus and cytoplasm. The La protein functions by protecting its small RNA ligands from exo-nucleolytic decay and also by serving as chaperone during folding. In addition to its major products (tRNAs and 5S rRNA), RNAP III synthesizes certain other non-coding RNAs. La-related protein-4 (LARP4) interacts with certain mRNAs and contributes to translational control and the cell’s growth capacity. Somewhat similar to La, LARP4 interacts with the 3′ end regions of its target RNAs, but in this case directed to the poly(A) motif (Yang et al., 2011 Mol Cell Biol 31:542-556).

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 the 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 et al., Mol Cell Biol 2013;33:4900; Lamichhane et al., Mol Cell Biol 2013;33:2918), and during mammalian development, and how its deficiency leads to childhood metabolic disease. We are also investigating how differences in the copy number of tRNA genes, which we found does indeed vary among humans, can affect how the genetic code is deciphered. Tumor suppressors and oncogenes mediate deregulation of transcript production by Pol III, the RNA polymerase 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, TRIT1, and LARP4 and their contribution 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

Recent findings regarding nucleolar localization, cytoplasmic splicing, and retrograde transport indicate 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. Thus, it has 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 et al., Mol Cell Biol 2014;34:123-131).

To study Pol 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 Saccharomyces cerevisiae with respect to cell-cycle control, gene-promoter structure, and the complexity of pre–mRNA splicing. From sequence analysis of Pol III–transcribed genes, we predicted and then confirmed that Pol III termination–signal recognition in S. pombe would be more similar to human Pol III than it is for S. cerevisiae Pol III. Our system is based on tRNA–mediated suppression of a nonsense codon in 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 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 Pol III termination–associated Pol 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 recent work suggest 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 et al., Nat Struct Mol Biol 2006;13:611; Maraia and Bayfield, 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 surface to promote a previously unknown step in tRNA maturation. One of our objectives is to identify cellular genes other than 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 and associated factors

The RNA polymerase III (RNAP III, Pol 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 Pol III to the correct start site. Pol 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 and Maraia, Mol Cell Biol 2013;33:1571).

Transcription termination delineates 3′ ends of gene transcripts, prevents otherwise runaway RNA polymerase (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 (Reference 5).

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 the RNAs’ common UUU-3′ OH–terminal elements; and (2) by unknown mechanisms, translation of specific subsets of mRNAs, such as those containing iron-response elements (IRES) and other motifs. The La-related protein LARP7/PIP7S exhibits a specialized UUU-3′ OH–related function in its specific interaction with 7SK snRNA. Another La-related protein, LARP4, is conserved in metazoa and, in accordance with experimental data we obtained, appears to be a translation factor. Unlike La and LARP7, LARP4 localizes to the cytoplasm, as demonstrated by immunofluorescence, and contains a highly conserved sequence similar to but a variant of the poly-A binding protein (PABP)–interaction motif-2 (PAM2) consensus found in other translation factors, including Paip1 and Paip2. PABP co-immunoprecipitates with Flag-LARP4 (F-LARP4) from human cells in an RNase–insensitive manner, while substitution of two key residues in the variant PAM2 consensus reduces PABP co-immunoprecipitation. F-LARP4 specifically co-immunoprecipitates with two other translation factors that we examined—elF4G and RACK1—although the interactions are sensitive to RNase. Antibodies to LARP4 showed that native endogenous LARP4 is cytoplasmic, co-immunoprecipitates with PABP in an RNase–insensitive manner, and co-sediments with the 40S subunit peak and polysomes; however, the peak shifts upon puromycin treatment to one indicating a smaller size than the 40S mRNP. Luciferase translation reporter assays in control and siRNA LARP4 knockdown cells provided evidence that LARP4 promotes general translation. The ability of LARP4 to stimulate translation of the luciferase reporter is correlated with its ability to stabilize mRNA levels. Indeed, actinomycin D studies show that LARP4 is an mRNA–stabilizing factor. Additional assays developed to better examine decay indicate a role for LARP4 in mRNA stability.

Fission yeast as a model system in which to study pathways of rapamycin sensitivity caused by defects in tRNA metabolism

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 C. 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.

Additional Funding

• NICHD Director's Intramural Research Program Award

Publications

1. Yarham JW, Lamichhane TN, Pyle A, Mattijssen S, Baruffini E, Bruni F, Donnini C, Vassilev A, He L, Blakely EL, Griffin H, Santibanez-Koref M, Bindoff LA, Ferrero I, Chinnery PF, McFarland R, Maraia RJ, Taylor RW. Defective i6A37 modification of mitochondrial and cytosolic tRNAs results from pathogenic mutations in TRIT1 and its substrate tRNA. PLoS Genet 2014; 10:e1004424.
2. Maraia RJ, Iben JR. Different types of secondary information in the genetic code. RNA 2014; 20:977-984.
3. Arimbasseri AG, Kassavetis GA, Maraia RJ. Transcription. Comment on “Mechanism of eukaryotic RNA polymerase III transcription termination.” Science 2014; 345:524.
4. Iben JR, Maraia RJ. tRNA gene copy number variation in humans. Gene 2014; 536:376-384.
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.

Collaborators

• David Clark, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
• Robert Crouch, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
• Markus Hafner, PhD, Laboratory of Muscle Stem Cells and Gene Regulation, NIAMS, Bethesda, MD
• Herbert C. Morse, III, MD, Laboratory of Immunogenetics, NIAID, Rockville, MD
• Peter R. Williamson, MD, PhD, Laboratory of Clinical Infectious Diseases, NIAID, Bethesda, MD

Contact

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

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