SEGR | SHCG | SMCB | SMGI | UCGE | Main Page

 

LA ANTIGEN AND ASSOCIATED RNA METABOLISM IN CELL BIOLOGY, GROWTH, AND DEVELOPMENT

 

Richard J. Maraia, MD, Head, Section on Molecular and Cell Biology

Robert V. Intine, PhD, Staff Scientist

Ying Huang, PhD, Visiting Associate

Mark Bayfield, PhD, Visiting Fellow

Elena Schwartz, PhD, Visiting Fellow

Stacy Kentner, BS, Technician

Monique Bruinsma, BS, Postbaccalaureate Fellow

Trish Kaiser, BS, Postbaccalaureate Fellow

Julie Mazeika, BS, Technical Training Fellow

 

Pol III transcript production supports cellular growth and proliferation because tRNAs and 5S rRNA facilitate protein synthesis. The La antigen is a eukaryote-ubiquitous RNA-binding protein that promotes maturation of tRNAs and other pol III transcripts. La facilitates a connection between transcription and post-transcriptional processing by binding to UUUOH, the 3´ motif that results from transcription termination of all genes transcribed by pol III. While the phospho-isoform of La promotes tRNA production, the nonphospho-isoform associates with abundant mRNAs that bear 5´ terminal oligopyrimidine (5´TOP), which produces ribosomal proteins and translation factors as well as snoRNAs involved in large rRNA biogenesis in the nucleolus. The short basic motif (SBM), a cis element in La, directs interaction with nucleolin in the nucleolus in a nonphosphorylation-specific manner, suggesting yet additional involvement in large rRNA maturation in the nucleolus. La may be involved in the metabolism of transcripts synthesized by all three nuclear RNA polymerases in a manner that contributes to a major central activity, the translational capacity of the cell. To investigate these issues, we use modern genetics, molecular biology, and biochemical approaches, relying heavily on analytic biochemistry as well as on tissue culture, yeast systems, and transgenic and gene-altered mice.

Functions of the human La antigen in RNA expression

Intine, Schwartz, Bayfield, Kaiser, Maraia

Human La antigen is a nuclear phosphoprotein that has so far been found in all cells of all of eukaryotes. As alluded to above, La protein is a target “antigen” of antibodies (Ab) in patients suffering from autoimmune disorders such as systemic lupus erythematosous (SLE, lupus), neonatal lupus, and Sjögren’s syndrome. Evidence from our and other laboratories indicates that La is a component of a pol III holoenzyme that remains associated with newly synthesized transcripts to direct their maturation. As such, La is considered a regulatory chaperone for nascent RNAs, as it can control their nuclear residence and accessibility to the processing enzymes. La carries out its regulatory function in part by sequence-specific binding to a UUU-OH 3´-terminal motif that is common to all transcripts synthesized by pol III and that results from transcription termination by RNA pol III.

We mapped the major phosphorylation site of La to serine 366 (S366) and showed that the phosphorylation interferes with La’s ability both to interact with the initiating pppG of the nascent transcript and to activate transcription initiation by Pol III, suggesting that such activities are mechanistically related. The implication is that an “La cycle” of transcription and post-transcriptional regulation is mediated in part by the C-terminal domain (CTD) of La. Others have recently shown that La is dephosphorylated at S366 early during apoptosis. We have developed two sets of monospecific antibodies that differ in their ability to recognize phosphoS366 (pLa) or nonphospohoS366 (npLa). The antibodies demonstrate that npLa and pLa exhibit different subnuclear localizations (nucleoplasm versus nucleoli) and that they are differentially associated with certain RNAs in vivo.

Our data indicate several trafficking signals in La that control nuclear, nucleolar, and cytoplasmic localization. Using a tRNA suppressor reporter system, we identified an intranuclear trafficking defect that is associated with detrimental disordering of the tRNA processing activities. In this case, La recognizes and binds normally to its substrate pre-tRNAs but is deficient for proper routing in the nucleus, which causes the accumulation of a dead-end tRNA processing intermediate that is nonfunctional.

Our studies not only revealed that La trafficks through the nucleus and is exported to the cytoplasm, but they also uncovered an unexpected nuclear export signal (NES) in La. An efficient, carrier-mediated NES system for La proved surprising because the established functions of La in pol III transcript biogenesis are entirely intranuclear. Further characterization revealed that the nuclear export pathway used for La and other features of its NES function are typical of some RNA binding proteins that carry certain mRNAs to the cytoplasm. Accordingly, we have begun to extend our studies to include examination of the involvement of La in the expression of certain target mRNAs, especially those whose expression is critical to growth and development.

Intine RV, Dundr M, Misteli T, Maraia RJ. Aberrant nuclear trafficking of La protein leads to disordered processing of associated precursor tRNAs. Mol Cell 2002;9:1113-1123.

Intine RV, Dundr M, Vassilev A, Zhou Y, DePamphilis ML, Maraia R. 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. Mol 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.

Trotta R, Vignudelli T, Candini O, Intine RV, Pecorari L, Guerzoni C, Santilli G, Byrom MW, Goldoni S, Ford LP, Caligiuri MA, Maraia RJ, Perrotti D, Calabretta B. BCR/ABL activates mdm2 mRNA translation via the La antigen. Cancer Cell 2003;13:145-160.

Transcription termination by RNA polymerase III

Huang, Mazeika, Maraia

As indicated in the above section, La protein binds to UUU-OH, the 3´ terminal motif of nascent transcripts, which results from termination by RNA pol III. We developed a pol III transcription system in the fission yeast S. pombe. A pol III-dependent gene encodes an opal suppressor tRNA that suppresses a nonsense codon in the mRNA encoding a purine-synthetic enzyme (Ade6-704), whose activity can be monitored by an in vivo colorimetric plate assay. We demonstrated that the expression of the gene in S. pombe is dependent on accurate and efficient termination by pol III and established that the minimal number of dT residues required for efficient termination is five. We have uncovered an intriguing correlation between sensitivity of RNA pol III to the toxin alpha-amanitin and to the pol III termination signal and have begun a structure-function analysis of the largest subunit of pol III by using our in vivo reporter system.

We have recently shown that, in fission yeast, mutations in the pol III subunit Rpc11p that decrease RNA 3´ cleavage activity increase 3´ oligo-U length and La-dependent tRNA processing. Termination by RNA polymerase (pol) III produces RNAs whose 3´ oligo-U termini are bound by La protein, a chaperone that protects RNAs from 3´ exonucleases and promotes RNA maturation. Several reports indicate that yeast uses La-dependent and La-independent pathways for tRNA maturation, with defective pre–tRNAs most sensitive to decay and dependent on La. The Rpc11p subunit of pol III is homologous to the zinc ribbon of TFIIS and was known to mediate RNA 3´ cleavage and to be important for termination. To screen S. pombe for rpc11-mutants that increase tRNA-mediated suppression, we used tRNASerUGAM, an opal suppressor that suppresses ade6-704 and the accumulation of red pigment. Analysis of two zinc ribbon mutants indicates that they 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 that contain an insufficient number of 3´ Us for efficient La binding appear to decay in wild-type cells but are processed along the La-dependent pathway in the mutants. The data indicate that Rpc11p can limit RNA 3´ oligo-U length and the La-dependent pathway of tRNA maturation in fission yeast. Our results thus suggest 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 as the nature of the mechanistic 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 whether other factors contribute to the La-dependent activation of this tRNA gene.

Huang Y, Hamada M, Maraia RJ. RNA polymerase III from the fission yeast, Schizosaccharomyces pombe. In: Adhya S, Garges V, eds. Methods in Enzymology: RNA Polymerases and Associated Factors. Parts C & D. San Diego: Academic Press, 2003;370-371.

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, in press.

Role of La antigen in mouse development

Kentner, Bruinsma, Maraia

La is an abundant RNA-binding protein whose various phospho-isoforms are associated with different classes of RNAs in different subcellular compartments. Nuclear La is associated with precursors of tRNAs and rRNAs while cytoplasmic La is associated with mRNAs that encode ribsomal proteins and general translation factors, consistent with a role in coordinating the production of the translational machinery. We have examined La expression during normal mouse development and created an La gene knockout mouse. A quantitative method for mRNA expression shows that La mRNA is present in the oocyte and egg, with a slight increase after fertilization and a decline thereafter through the eight-cell stage. Immunostaining of whole blastocysts shows that La protein is readily detectable in nuclei of day 4.5 blastocysts. By embryonic stage day seven, La mRNA is abundant. No homozygous La–/– embryos survived to birth. The ratio of La+/– to La+/+ mice from heterozygous intercrosses was 1.7:1, suggesting that a fraction of heterozygotes do not survive to birth, although the surviving La+/– mice had no recognizable deleterious phenotype. Northern analysis revealed that La mRNA levels in heterozygous La+/− mice were reduced to 65 percent of La+/+ mice. Nullizygous La–/– blastocysts were recovered in expected numbers before implantation, but not at E6.0 or later, indicating that La is required for early development. The data are consistent with a maternal store of La mRNA, with La protein detection in day 4.5 blastocysts, as well as with our data indicating an essential requirement for La during early development. Mouse La nullizygous ES cells are under development.

COLLABORATORS

Bruno Calabetta, MD, PhD, Kimmel Cancer Center, Jefferson University, Philadelphia, PA

Melvin DePamphilis, PhD, Laboratory of Molecular Growth Regulation, NICHD, Bethesda, MD

Jack Keene, PhD, Duke University Medical Center, Durham, NC

Danillo Perrotti, MD, PhD, Kimmel Cancer Center, Jefferson University, Philadelphia, PA

Scott Tenenbaum, PhD, Duke University Medical Center, Durham, NC


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