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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
- Tek Lamichhane, PhD, Visiting Fellow
- Joowon Lee, PhD, Visiting Fellow
- Sandy Mattijssen, PhD, Visiting Fellow
- Keshab Rijal, PhD, Visiting Fellow
We are interested in how the biogenesis and metabolism pathways for RNAs, especially tRNAs and mRNAs, intersect with pathways related to cell proliferation, growth, and development. We focus on tRNA synthesis by RNA polymerase (Pol) III and its post-transcriptional handling by the RNA–binding protein La. Together with La-related protein-4 (LARP4), La interacts with certain mRNAs and contributes to translational control and the cell’s growth capacity. 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.
In addition to its major products (tRNAs and 5S rRNA), Pol III synthesizes other non-coding RNAs. We focus on the tRNA–modification enzyme tRNA isopentenyltransferase (TRIT1) and the effects of its modifications on tRNA activity in translation in a codon-specific manner. We are also investigating how variances in the copy number of tRNA genes, which we have found variies among humans, can affect how the genetic code is deciphered. Tumor suppressors and oncogenes mediate deregulation of Pol III transcript production, contributing to increased capacity for proliferation of cancer cells.
We 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.
Red-white colony differentiation by tRNA–mediated suppression
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 in 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: (i) the human pattern of phosphorylation of hLa on the CK2 target site serine-366 occurs faithfully in S. pombe and promotes tRNA production; (ii) various conserved subcellular trafficking signals in La proteins can be positive or negative determinants of tRNA processing; (iii) La can protect pre–tRNAs from the nuclear surveillance 3′ exonuclease Rrp6p; (iv) the 3′ exonuclease that processes pre–tRNAs in the absence of Sla1p is distinct from Rrp6p; (v) 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 (vi) 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 Pol III enzyme consists of 17 subunits, several with strong homology to subunits of Pol I and Pol 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 Pol I, Pol II, and Pol 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, Pol II must be highly processive and avoid premature termination. Pol 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 Pol III transcripts appears to occur at the Pol III active center. The dT(n) tracts at the ends of class III genes directly signal pausing and release by Pol III such that termination and RNA 3′ end formation are coincident and efficient.
Rpc11p is an integral Pol III subunit that mediates conserved exo-ribonucleolytic cleavage of the nascent RNA 3′ end within the Pol III transcription complex. Accumulated data suggest that Rpc11p is involved in termination and efficient recycling of Pol III. We showed that mutations impairing Rpc11p’s RNA 3′ cleavage activity alter RNA 3′ end formation in vivo with consequences for tRNA production. Given that Rpc11p has homologs in Pol II (Rpb9p and TFIIS) and Pol I (Rpa11p), we suspect that Rpc11p’s mechanism of action may also operate in these homologs. Impairment of a conserved interaction between Rpc11p and the core—Rpc2p (second-largest Pol III subunit)—leads to tissue-specific defects in zebrafish development. As we discovered, the pol III subunit Rpc37 is a potent source of mutations that impair the ability of pol III to recognize or pause at the pol III termination signal.
La-related protein-4 (LARP4) in translation-coupled mRNA stabilization
The eukaryote-ubiquitous La proteins are involved in two broad functions: (i) 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 (ii) translation of specific subsets of mRNAs, such as those containing iron-response elements (IRES) and other motifs, by unknown mechanisms. 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 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 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 gene tit1+, which encodes tRNA isopentenyl transferase, causes S. pombe proliferation to become sensitive to rapamycin, with a "wee" phenotype, suggesting a cell cycle defect. 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. tit1Δ cells exhibit anti-suppression, indicating a requirement for i6A37 for optimal codon-specific translation efficiency, as well as defects in carbon metabolism related to respiration. 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 tit1Δ 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 Tit1p–modified tRNA Tyr exhibits about five-fold higher specific decoding activity during translation than the unmodified tRNA Tyr.
- Gaidamakov S, Maximova OA, Chon H, Blewett NH, Wang H, Crawford AK, Day A, Tulchin N, Crouch RJ, Morse HC, Blitzer RD, Maraia RJ. Targeted deletion of the gene encoding the La protein SSB autoantigen in B cells or cerebral cortex causes extensive tissue loss. Mol Cell Biol 2013;in press.
- Lamichhane TN, Mattijssen S, Maraia RJ. Human cells have a limited set of tRNA anticodon loop substrates of the tRNA isopentenyltransferase TRIT1 tumor suppressor. Mol Cell Biol 2013;Epub ahead of print.
- Lamichhane TN, Blewett NH, Crawford AK, Cherkasova VA, Iben JR, Begley TJ, Farabaugh PJ, Maraia RJ. Lack of tRNA modification isopentenyl-A37 alters mRNA decoding and causes metabolic deficiencies in fission yeast. Mol Cell Biol 2013;33:2918-2929.
- Arimbasseri AG, Maraia RJ. Distinguishing core and holoenzyme mechanisms of transcription termination by RNA polymerase III. Mol Cell Biol 2013;33:1571-1581.
- Cherkasova V, Maury LL, Bacikova D, Pridham K, Bähler J, Maraia RJ. Altered nuclear tRNA metabolism in La-deleted Schizosaccharomyces pombe is accompanied by a nutritional stress response involving Atf1p and Pcr1p that is suppressible by Xpo-t/Los1p. Mol Biol Cell 2012;23:480-491.
- Robert D. Blitzer, PhD, Icahn Medical Institute, Mount Sinai Hospital, New York, NY
- David Clark, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
- Robert Crouch, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
- Herbert C. Morse, III, MD, Laboratory of Immunogenetics, NIAID, Rockville, MD