The La antigen associates with all transcripts synthesized by RNA polymerase (pol) III and binds to these transcripts by high-affinity recognition of their common 3´ terminal motif, UUU-OH, which results from transcriptional termination by pol III. RNA production by pol III is required to support growth and development; specifically, the transcripts tRNAs, 5S rRNA, and other small noncoding RNAs are required for protein synthesis and other vital cellular functions. The human La antigen is a target of autoimmune antibodies in patients suffering from systemic lupus erythematosus, neonatal lupus, and Sjögren’s syndrome. Although La proteins are ubiquitous, abundant, and multifunctional in eukaryotes, they have been best characterized in human and yeast cells. They associate with transcripts ranging from tRNAs and other small noncoding RNAs to subsets of growth-related mRNAs. In the nucleus, La promotes the maturation of small RNAs, including tRNA precursors, in part by affecting the order in which the 5´ and 3´ ends and introns are processed and in part by retaining the precursor RNAs in close proximity to processing enzymes in the nucleus. In the cytoplasm, La facilitates the translation initiation of mRNAs. Thus, La is involved in controlling several aspects of RNA metabolism.
Functions of the human La antigen in RNA expression
Intine, Schwartz, Bayfield, Kaiser, Maraia; in collaboration with White
While the phosphoisoform of La promotes tRNA maturation in the nucleus, the nonphosphoisoform found in the cytoplasm associates with mRNAs that bear the 5´-terminal oligopyrimidine (5´TOP) motif and produce ribosomal proteins and translation factors as well as snoRNAs involved in large rRNA biogenesis in the nucleolus. A cis element in La, the short basic motif (SBM), directs interaction with nucleolin in the nucleolus in a non–phosphorylation-specific manner, suggesting yet other involvement in large rRNA maturation in the nucleolus. As such, La may be involved in the metabolism of transcripts synthesized by all three nuclear RNA polymerases and, moreover, in a manner that contributes to the cell’s translational capacity.
Evidence from our and other laboratories indicates that La is a component of a Pol III holoenzyme that associates with newly synthesized transcripts to direct their maturation. Thus, given that it can control nuclear residence of and accessibility to the processing enzymes, La is considered a regulatory chaperone for nascent RNAs. While the results from our laboratory and others have indicated a role for La in transcription by pol III, the results of still other laboratories do not support our conclusion.
We mapped the major phosphorylation site of La to serine 366 (S366) and showed that the phosphorylation inhibits La’s ability to activate transcription by Pol III. The results suggest an “La cycle” of transcription and post-transcriptional regulation. We developed two sets of monospecific antibodies that differ in their ability to recognize phosphoS366 (pLa) or nonphospohoS366 (npLa) and used them to demonstrate that npLa and pLa exhibit distinct subnuclear localizations (nucleoplasm versus nucleoli and cytoplasm) and that they are differentially associated with certain RNAs in vivo. In collaboration with Robert White, we recently showed that the nonphosphoisoform of La (rather than the phosphoisoform) is found at the tRNA and other pol III-transcribed genes in vivo.
Our cell-biological investigations indicated several trafficking signals in La that control nuclear, nucleolar, and cytoplasmic localization. Using a tRNA suppressor reporter system in yeast, we identified an intranuclear trafficking defect that is associated with disordering of the steps in the tRNA processing pathway. In this case, La recognizes and binds to its substrate pre–tRNAs normally but is deficient for proper routing in the nucleus, thereby causing the accumulation of a dead-end tRNA processing intermediate that is nonfunctional. Given that La is conserved, we are able to use our yeast system to make advances in our research on human La and yeast La protein.
Our studies revealed that La traffics extensively; we also uncovered functional nuclear export activity that is conserved in La proteins from yeasts to humans. The latter discovery was surprising because the established functions of La in Pol III transcript biogenesis are entirely intranuclear. Characterization revealed that the pathway and other features of La’s nuclear export activity are reminiscent of RNA binding proteins that carry mRNAs to the cytoplasm. Accordingly, we have begun to extend our studies to include the involvement of La in the expression of certain target mRNAs, especially those whose expression is critical to growth and development.
Fairley JA, Kantidakis T, Kenneth NS, Intine RV, Richard Maraia R, White RJ. Human La is found at RNA polymerase III-transcribed genes in vivo. Proc Natl Acad Sci USA (in press).
Intine RV, Dundr M, Vassilev A, Schwartz E, Zhao Y, DePamphilis ML, Maraia RJ. Nonphosphorylated human La antigen interacts with nucleolin at nucleolar sites involved in rRNA biogenesis. Mol Cell Biol 2004;24:10894-10904.
Intine RV, Tenenbaum SA, Sakulich AS, Keene JD, Maraia RJ. Differential phosphorylation and subcellular localization of La RNPs associated with precursor tRNAs and translation-related mRNAs. Molecular Cell 2003;12:1301-1307.
Schwartz E, Intine RV, Maraia RJ. CK2 is responsible for phosphorylation of human La protein serine-366 and can modulate 5´TOP mRNA metabolism. Mol Cell Biol 2004;24:9580-9591.
Transcription termination by RNA polymerase III
Huang, Mazeika, Maraia
As indicated above, La protein binds to the 3´ UUU-OH motif that results from termination by RNA Pol III. We used a model transcription system in the fission yeast Saccharomyces pombe that relies on an opal suppressor tRNA (tRNASerUGAM) to suppress a nonsense codon in the mRNA encoding a purine-synthetic enzyme (Ade6-704); the enzyme’s activity can be monitored by an in vivo colorimetric colony assay. The expression of this tRNA gene is dependent on accurate and efficient termination by pol III, which requires a minimum of five of dT residues.
In fission yeast, mutations in the polymerase III subunit Rpc11p that decrease RNA 3´ cleavage activity increase 3´ oligo-U length and La-dependent tRNA processing. Rpc11p is highly homologous to the zinc ribbon of TFIIS and known to mediate RNA 3´ cleavage. We used the tRNA suppressor and our red/white colony assay to screen S. pombe for rpc11 mutants that increase tRNA-mediated suppression. Analysis of two zinc ribbon mutants indicated that the mutants are deficient in pol III–associated RNA 3´ cleavage activity and produce pre–tRNAs with elongated 3´ oligo-U tracts that are better substrates for La-dependent processing. We found that a substantial fraction of pre–tRNASerUGAM, which contains an insufficient number of 3´ U nucleotides for efficient La binding, appears to decay in wild-type cells while the mutants are processed along the La-dependent pathway. The data indicate that, in fission yeast, Rpc11p can limit the RNA 3´ oligo-U length and the La-dependent pathway of tRNA maturation, suggesting that a novel distinct step in pol III termination is the shortening of the 3´ oligo-U of newly synthesized transcripts in a manner that affects their post-transcriptional processing. The data also suggest that Rpc11p may be considered a quality control factor for tRNA production.
Many important questions remain, such the nature of the link between La and pol III termination; whether the lack of reporter gene–derived transcripts in the La-minus strain is attributable to a defect in transcription rate, nascent RNA processing, or both; and what other factors contribute to the La-dependent activation of the tRNA gene.
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.
Role of La antigen in mouse development
Park, Kaiser, Bruinsma, Maraia
Ubiquitous in eukaryotes, La functions as an RNA binding protein that promotes the maturation of tRNA precursors and other small noncoding RNAs. In addition, it associates with mRNAs that encode ribosome subunits and precursors to snoRNAs involved in ribosome biogenesis. Thus, it was surprising that La is dispensable in yeasts, the organisms in which it has been most extensively characterized. To determine if La is essential in mammals and, if so, at which developmental stage it is required, we created mice with a disrupted La gene and analyzed the offspring from La+/– / La+/– intercrosses. We detected La–/– offspring at the expected frequency among blastocysts before uterine implantation but no nullizygotes after implantation, indicating that La is required early in development. We obtained La+/– mice at 25 percent less than the expected frequency, which suggested that La haploinsufficiency may be detrimental in some genetic backgrounds. Blastocysts derived from La+/– / La+/– intercrosses yielded 38 La+/+ and La+/– embryonic stem (ES) cell lines but no La–/– ES cell lines, indicating that La plays a critical role in establishing ES cells. Consistent with this finding, blastocyst outgrowth assays revealed loss of the inner cell mass specifically from La–/– embryos. The results suggest that La is essential in mammals, in contrast to yeasts, and is one of a limited number of genes required as early as development of the inner cell mass.
Park JM, Kohn MJ, Bruinsma M, Vech C, Intine RV, Grinberg SF, Mukherjee A, Love PI, Ko MS, DePamphilis ML, Maraia RJ. The multifunctional RNA-binding protein La antigen is required for mouse development and for the establishment of embryonic stem cells. Mol Cell Biol 2005 (in press).
COLLABORATORS
Mel DePamphilis, PhD, Laboratory of Molecular Growth Regulation, NICHD, Bethesda, MD
Minoru Ko, PhD, Laboratory of Genetics, NIA, Baltimore, MD
Paul Love, PhD, Laboratory of Mammalian Genes and Development, NICHD, Bethesda, MD
Robert White, PhD, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK
For further information, contact maraiar@mail.nih.gov.