LA ANTIGEN, RNA POLYMERASE III, AND ASSOCIATED RNA METABOLISM IN CELL BIOLOGY, GROWTH, AND DEVELOPMENT
, Head, Section on Molecular and Cellular Biology
Vera Cherkasova, PhD, Senior Fellow
James Iben, PhD, Research Fellow
Ruiqing Yang, PhD, Research Fellow
Mark A. Bayfield, PhD, Visiting Fellow
Liqiang Zhang, PhD, Visiting Fellow
Nathan H. Blewett, BS, Postbaccalaureate Fellow
Amanda M. Day, BS, Postbaccalaureate Fellow
Abduraman Elkatali, BS, Postbaccalaureate Fellow
Dagmar Bacikova, MS, Technician
We are interested in how the pathways of tRNA and other small 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 proteins (LRPs), contributes to ribosome production and the cell’s growth capacity. In addition to its major products, tRNAs and 5S rRNA, Pol III synthesizes other noncoding RNAs. Deregulation of Pol III transcript production is mediated by tumor suppressors and oncogenes and contributes to an increased capacity for proliferation in cancer cells. La protein is a target of autoantibodies that are prevalent (and diagnostic) in patients suffering from Sjögren’s syndrome, systemic lupus, and neonatal lupus. La comprises several nucleic acid binding motifs as well as several subcellular trafficking signals and associates with noncoding and messenger RNAs to coordinate activities in the nucleus and cytoplasm. Our goal is to understand the structure-function relationship and cell biology of La’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
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 to prevent their premature export, and promotion of a newly identified processing step distinct from 3¢-end protection. We had previously developed a red-white reporter system in the fission yeast S. pombe to study Pol III– and La-dependent tRNA biogenesis. We chose S. pombe as a model system because, in general, it appears more similar to human than S. cerevisiae in cell-cycle control, gene promoter structure, and 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 is more similar to human Pol III than is S. cerevisiae Pol III. Our system is based on tRNA-mediated suppression (TMS) of a nonsense codon in ade6-704 and affords the benefits of fission yeast biology while lending itself in certain aspects to “humanization.” We have been able to study the tRNA processing–associated function of the human La protein (hLa), recognizing that the function is so highly conserved that it can replace the same 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, which 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 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 for different functions. Consistent with this notion, some pre–tRNAs require only the UUU-3¢OH binding activity while others require a second activity in addition to 3¢ end protection; that second activity involves 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 the 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;27:3303-12.
Huang Y, Bayfield MA, Intine RV, Maraia RJ. Separate RNA-binding surfaces on the multifunctional La protein mediate distinguishable activities in tRNA maturation. Nat Struct Mol Biol 2006;13:611-8.
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;25:621-36.
Maraia RJ, Bayfield MA. The La protein-RNA complex surfaces. Mol Cell 2006;21:149-52.
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, transcription factor 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 support many cycles of initiation, termination, and re-initiation with high productivity. 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. Because 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 requiring endonucleolytic cleavage of the 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 a conserved exoribonucleolytic cleavage of the nascent RNA 3¢ end within the Pol III transcription complex. Cumulative data suggest that Rpc11p is involved in termination and efficient recycling of Pol III. We showed that mutations that impair the RNA 3¢ cleavage activity of Rpc11p alter RNA 3¢ end formation in vivo with consequences for tRNA production. Given Rpc11p’s homologues in Pol II (Rpb9p and TFIIS) and Pol I (Rpa11p), we suspect that understanding Rpc11p’s mechanism of action may be applicable to the latter enzymes.
Our collaborator Michael Pack made an exciting discovery related to Rpc11p. Impairment of a conserved interaction between Rpc11p and the core, second-largest Pol III subunit Rpc2p 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 less proliferative cells of the developing organism. 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.
In collaboration with Shiv Grewal, we have shown that the Sfc3p and Sfc6p subunits of S. pombe TFIIIC provide insight into genome organization. In particular, 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.
Fairley JA, Kantidakis T, Kenneth NS, Intine RV, Maraia RJ, White RJ. Human La is found at RNA polymerase III-transcribed genes in vivo. Proc Nat Acad Sci USA 2005;102:18350-5.
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;25:621-36.
Noma K, Cam HP, Maraia RJ, Grewal SIS. A novel function for TFIIIC transcription factor complex in genome organization. Cell 2006;125:859-72.
Yee N, Gong W, Huang Y, Lorent K, Dolan A, Maraia R, Pack M. Mutation of RNA polymerase III subunit rpc2/polr3b leads to deficiency of the RNA cleavage subunit, Rpc11/Polr3k, and disrupts zebrafish digestive system development. PLOS Biol 2007;5:e312.
Role of La antigen in mouse development
As noted above, La protein is a target of autoantibodies in patients suffering from Sjögren’s syndrome, systemic lupus erythematosus, and neonatal lupus. Ubiquitous in eukaryotic nuclei, the La phosphoprotein functions to promote the maturation of tRNA precursors and other small noncoding RNAs synthesized by RNA Pol III. In the cytoplasm, the nonphosphorylated isoform of human La is associated with a family of mRNAs that bear a common 5¢ terminal oligopryimidine (5¢ TOP) motif; that family of mRNAs coordinately produces ribosomal proteins and translation factors in response to nutritional status. Thus, it was surprising that La is dispensable in yeasts, the organisms in which it has been characterized most extensively. To determine if La is essential in mammals and, if so, at what developmental stage it is required, we generated mice with a disrupted La gene and analyzed the offspring from La+/− X La+/− intercrosses. We detected La−/− offspring at the expected frequency among blastocysts before uterine implantation but observed no nullizygotes after implantation, indicating that La is required early in development.
Blastocysts derived from La+/− X La+/− intercrosses yielded 38 La+/+ and La+/− embryonic stem (ES) cell lines but no La−/− ES cell lines, suggesting that La contributes a critical function in the establishment of ES cells. Consistent with this notion, blastocyst outgrowth assays revealed loss of the inner cell mass specifically from La−/− embryos. The results indicate that, in contrast to yeasts, La is essential in mammals and is one of a limited number of genes required as early as development of the inner cell mass.
We have now generated conditional knockout alleles of La and are investigating the resulting mice by using appropriate Cre-mater mice. One goal is to determine at what developmental stage, if any, La can be deleted with minimal effects on development. Another goal is to obtain ES cell lines that can be used to examine the effects of loss of the conditional La knockout allele.
Park JM, Kohn MJ, Bruinsma MW, Vech C, Intine RV, Fuhrmann S, Grinberg A, Mukherjee I, Love PE, Ko MS, DePamphilis ML, Maraia RJ. The multifunctional RNA-binding protein La is required for mouse development and for the establishment of embryonic stem cells. Mol Cell Biol 2006;26:1445-51.
1 Robert V. Intine, PhD, former Staff Scientist
2 Ying Huang, PhD, former Visiting Associate
3 Trish E. Kaiser, BS, former Technician
4 Jung-Min Park, PhD, former Visiting Fellow
5 Monique W. Bruinsma, BS, former Postbaccalaureate Fellow
COLLABORATORS
Jurg Bahler, PhD, Wellcome Trust Sanger Institute, Cambridge, UK
Sailen Barik, PhD, University of South Alabama College of Medicine, Mobile, AL
Melvin DePamphilis, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
Shiv Grewal, PhD, Laboratory of Molecular Cell Biology, NCI, Bethesda, MD
Minoru Ko, PhD, Laboratory of Genetics, NIA, Baltimore, MD
Paul Love, MD, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
Michael Pack, MD, University of Pennsylvania School of Medicine, Philadelphia, PA
Scott Tenenbaum, PhD, University at Albany-SUNY, Albany, NY
Alex Vassilev, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
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.
