La Antigen, RNA Polymerase II, and Associated RNA Metabolism In Cell Biology, Growth, and Development

Richard J. Maraia, MD, Head, Section on Molecular and Cellular Biology
Robert V. Intine, PhD, Staff Scientist¹
James Iben, PhD, Research Fellow
Ruiqing Yang, PhD, Research Fellow
Vera Cherkasova, PhD, Senior Fellow
Mark A. Bayfield, PhD, Visiting Fellow
Jung-Min Park, PhD, Visiting Fellow¹
Ying Huang, PhD, Visiting Associate¹
Jonathan Jacobs, PhD, Postdoctoral Fellow
Dagmar Bacikova, MS, Technician
Nathan H. Blewett, BS, Postbaccalaureate Fellow¹
Amanda K. Crawford, BS, Postbaccalaureate Fellow
Amanda M. Day, BS, Postbaccalaureate Fellow¹

We are interested in how the pathways of tRNA and other RNA biogenesis interact with pathways that control cell proliferation, growth, and development. We focus on RNA polymerase (Pol) III and the post-transcriptional handling of its transcripts by the RNA-binding protein La, which, together with La-related protein-4 (LARP4), contributes to ribosome production, translational control, and the cell’s growth capacity. In addition to its major products (tRNAs and 5S rRNA), Pol III synthesizes other non-coding RNAs. Tumor suppressors and oncogenes mediate deregulation of Pol III transcript production, contributing to increased capacity for proliferation of cancer cells. La protein is a target of autoantibodies prevalent (and diagnostic) in 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. We strive to understand the structure-function relationship and cell biology of La’s and LARP4’s 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

Maraia, Bayfield

Recent paradigm-shaking findings such as 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 pre–tRNAs’ premature export; and promotion of a newly identified processing step distinct from 3′-end protection.

To study Pol III- and La-dependent tRNA biogenesis, we had developed a red-white reporter system in the fission yeast S. pombe. That model system generally appears more similar to the human organism than does S. 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 (1) the human pattern of phosphorylation of hLa on the CK2 target site serine-366 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 RNA recognition motif-1 (RRM1), to promote distinct steps in tRNA maturation.

Our recent work suggests that La can use several surfaces, perhaps combinatorially, to engage different 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 have isolated and begun to characterize S. pombe revertant mutants that overcome a defect in the second activity.

Bayfield MA, Kaiser TE, Intine RV, Maraia RJ. Conservation of a masked nuclear export activity of La proteins and its effects on tRNA maturation. Mol Cell Biol 2007;27:3303-3312.
Bitko V, Musiyenko A, Bayfield MA, Maraia RJ, Barik S. Cellular La protein shields nonsegmented negative-strand RNA viral leader RNA from RIG-I and enhances virus growth by diverse mechanisms. J Virol 2008;82:7977-7987.
He N, Jahchan NS, Hong E, Li Q, Bayfield MA, Maraia RJ, Luo K, Zhou Q. A La-related protein modulates 7SK snRNP integrity to suppress P-TEFb-dependent transcriptional elongation and tumorigenesis. Mol Cell 2008;29:588-599.
Maraia RJ, Blewett NH, Bayfield MA. It’s a mod mod tRNA world. Nat Chem Biol 2008;4:162-164.
Park JM, Intine RV, Maraia RJ. Mouse and human La proteins differ in kinase substrate activity and activation mechanism for tRNA processing. Gene Expr 2007;14:71-81.

Activities of RNA polymerase III and associated factors

Maraia, Huang, Intine; in collaboration with Pack, White, Grewal

The Pol III enzyme consists of 17 subunits, several with strong homology to subunits of Pol I and Pol II. In addition, TFIIIC, composed of 6 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 unique functions related to the different types of genes they transcribe. Given that some mRNA genes can be hundreds of kilobasepairs long, Pol II must be highly processive and avoid premature termination. Pol II terminates in response to complex termination–RNA processing signals that require endonucleolytic 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 in 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 exoribonucleolytic 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 homologues in Pol II (Rpb9p and TFIIS) and Pol I (Rpa11p), we suspect that understanding Rpc11p’s mechanism of action may be applicable to these homologues.

Our collaborator Michael Pack made an exciting finding related to Rpc11p. Impairment of a conserved interaction between Rpc11p and the core—Rpc2p (second-largest Pol III subunit)—leads to tissue-specific defects in zebrafish development. A random genetic screen uncovered a small deletion in Rpc2p that impairs interaction with Rpc11p. The homologous deletion engineered in S. pombe Rpc2p impaired recruitment of Rpc11p by S. pombe Pol III. The zebrafish developmental defects manifest in highly proliferative cells while sparing the less proliferative cells of the developing embryo. Remarkably, the mutant phenotype caused by the small deletion in Rpc1p can be rescued by overexpression of Rpc11p in zebrafish embryos. The precise role of Rpc11p in tissue-specific development remains to be determined.

A collaboration with Shiv Grewal focusing on the Sfc3p and Sfc6p subunits of S. pombe TFIIIC has provided insight into genome organization, as these B box–associated Pol III transcription factors were found to contribute to the boundary elements that partition silenced heterochromatin domains to the nuclear periphery.

Huang Y, Intine RV, Mozlin A, Hasson S, Maraia RJ. Mutations in the RNA polymerase III subunit Rpc11p that decrease RNA 3′ cleavage activity increase 3′-terminal oligo(U) length and La-dependent tRNA processing. Mol Cell Biol 2005;25:621-636.
Noma KI, Cam1 HP, Maraia RJ, Grewal SIS. A novel function for TFIIIC transcription factor complex in genome organization. Cell 2006;125:859-872.
Yee NS, Gong W, Huang Y, Lorent K, Dolan AC, Maraia RJ, Pack M. Mutation of RNA Pol III subunit rpc2/polr3b leads to deficiency of subunit Rpc11 and disrupts zebrafish digestive development. PLoS Biol 2007;5:e312.

La-related protein-4 (LARP4) in translational control

Maraia, Yang

The eukaryote-ubiquitous 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) translation of specific subsets of mRNAs, such as those containing 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 criteria that 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.

LARP4 immunoprecipitation followed by microarray analysis identified target mRNAs, which we verified by RT-PCR for the best two candidates. LARP4 levels declined by about 90 percent in these cells, but not in the control siRNA cells, without lowering actin or GAPDH. Preliminary data indicate that the LARP4-associated mRNAs peak on heavy polysomes in the control siRNA cells but shift off heavy polysomes and overaccumulate in the 80S peak in the siRNA-LARP4 cells. Attempts to reproduce and expand on these studies and identify common sequences in the LARP4-associated mRNAs are under way.

¹Former member of the laboratory

Collaborators

Jurg Bahler, PhD, Wellcome Trust Sanger Institute, Cambridge, UK
Sailen Barik, PhD, University of South Alabama College of Medicine, Mobile, AL
Shiv Grewal, PhD, Laboratory of Molecular Cell Biology, NCI, Bethesda, MD
Michael Pack, MD, University of Pennsylvania School of Medicine, Philadelphia, PA
Scott Tenenbaum, PhD, University at Albany-SUNY, Albany, NY
Robert White, PhD, FRSE, FMedSci, University of Glasgow, Glasgow, UK
Qiang Zhou, PhD, University of California Berkeley, Berkeley, CA

For further information, contact maraiar@mail.nih.gov.