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We are interested in how the biogenesis and metabolism pathways for small RNAs, especially tRNAs and mRNAs, 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 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. 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 exonucleolytic decay. Our recent studies showed that human LARP4 protects certain mRNAs from decay, in a polyribosome-associated manner. 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
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 pre–tRNAs' premature export; and promotion of a newly identified processing step distinct from 3′-end protection.
Figure 1. The fission yeast Saccharomyces pombe as a model organism
Red-white colony differentiation by tRNA-mediated suppression
To study Pol III– and La-dependent tRNA biogenesis, we had developed a red-white tRNA–sensitive reporter system in the fission yeast S. pombe (Figure 1), a yeast that 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 (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 diifferent steps in tRNA maturation.
Our recent work suggests 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 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 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 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 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.
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 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 a mRNA–stabilizing factor.
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, suggestive of 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 antisuppression, 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 evidence that mRNAs enriched in codons cognate to i6A37-modified tRNAs are translated less efficiently than mRNAs with low content of the cognate codons.