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

Robert V. Intine, PhD, Staff Scientist
Ying Huang, PhD, Visiting Associate
Elena Schwartz, PhD, Visiting Fellow
Stacy Kentner, BS, Technician
Jessica Toher, BS, Postbaccalaureate Fellow
Sam Hasson, BS, Postbaccalaureate Fellow

Using transcription by RNA-polymerase III (Pol III) as a model system of gene expression, we study mechanisms of eukaryotic RNA biogenesis and functions of the human La antigen. The Pol III transcripts studied most intensely are the transfer RNAs. The precursors of tRNAs are synthesized as nascent transcripts that undergo a "maturation" process involving a series of coordinated enzymatic and intracellular trafficking activities that ultimately deliver a functional tRNA to the site of protein synthesis in the cytoplasm, specifically, the ribosomes. We focus on transcription initiation and termination as well as on RNA processing and intranuclear trafficking, some of the steps in RNA production, and how the sequential steps are coordinated. We have been especially interested in the human La antigen, a protein that is a target of autoantibodies in patients suffering from certain autoimmune disorders (e.g., systemic lupus erythematosus). The human La antigen is a nuclear-cytoplasmic shuttling phosphoprotein that also binds to the 5' untranslated regions of certain mRNAs and appears to regulate and coordinate critical steps in RNA expression. 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

Maraia, Intine, Schwartz, Treanor,a Vechb

Human La antigen is a nuclear phosphoprotein that so far has 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 in order 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 posttranscriptional 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 have identified an intranuclear trafficking defect that is associated with detrimental disordering of the tRNA processing activities. In this case, La recognizes and binds to its substrate pre-tRNAs normally but is deficient for proper routing in the nucleus, which causes the accumulation of a dead-end tRNA processing intermediate that is nonfunctional.

In revealing that La trafficks through the nucleus and is exported to the cytoplasm, our studies have uncovered an unexpected nuclear export signal (NES) in La. The finding of 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 as well as other features of its NES function are characteristic of some RNA binding proteins that carry certain mRNAs to the cytoplasm. Accordingly, we have begun to extend our studies to examine the involvement of La in the expression of certain target mRNAs, especially those whose expression is critical to growth and development.

Bhattacharya R, Perumal K, Sinha K, Maraia R, Reddy R. Methyl-phosphate cap structure in small

RNAs reduces the affinity of RNAs to La protein. Gene Expr 2002;10:243-253.

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.

Maraia RJ, Intine RV. La protein and its associated small nuclear and nucleolar precursor RNAs.

Gene Expr 2002;10:41-47.

Maraia RJ, Intine RV. Recognition of nascent RNA by the human La antigen: conserved and diverged

features of structure and function. Mol Cell Biol 2001;21:367-379.

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

Maraia, Huang, Mozlinc

As indicated above, La protein binds to the 3' terminal motif of nascent transcripts UUU-OH, which results from termination by RNA Pol III. We have 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 the sensitivity of RNA Pol III to both the toxin alpha-amanitin and the Pol III termination signal and, using our in vivo reporter system, have begun a structure-function analysis of the largest subunit of Pol III. We have shown that our tRNA gene requires the La protein for efficient expression in vivo. Many important questions remain, such as the mechanistic link between La and pol III termination; whether the lack of reporter gene-derived transcripts in the La-minus strain are 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.

Hamada M, Sakulich AL, Koduru SB, Maraia R. Transcription termination by RNA polymerase III in

fission yeast: a genetic and biochemically-tractable model system. J Biol Chem 2000;275:29076-29081.

Huang Y, Hamada M, Maraia RJ. RNA polymerase III from the fission yeast, Schizosaccharomyces

pombe. In: Adhya S, Garges S, eds. Methods in Enzymology RNA Polymerases and Associated Factors. Part C & D. San Diego: Academic Press, Inc.2003;370-371; in press.

Transcription initiation by RNA polymerase III

Maraia, Huang, McGillicuddyd

Although a longstanding interest of this section has been transcriptional termination, our recent discoveries have led us to examine mechanisms of Pol III initiation. Specifically, ongoing studies focus on the two central transcription factors (TF): TATA-binding protein (TBP) and the TFIIB-related factor Brf; both function at the core promoter. Homologs of these factors exist in all eukaryotes and archaea and are central in controlling transcription initiation. The core promoter is itself of central importance in transcription because, in addition to directing transcription initiation, core promoters integrate complex input from distal regulatory elements. Genomewide analysis has revealed that, in contrast to the core promoters of human and S. cerevisiae tRNA genes, which have long been known to be TATA-less, the core promoters of tRNA and 5S rRNA genes in S. pombe contain TATA elements. We used tRNA-dependent suppression and other in vivo assays as well as in vitro transcription to demonstrate an obligatory requirement for upstream TATA elements for tRNA and 5S rRNA expression in S. pombe. We found similar results for large rRNA synthesis, as mutation of the TATA element in the Pol I promoter also abolished rRNA expression in fission yeast, demonstrating TATA-unified transcription systems in eukaryotes and providing insight into the residual need for TBP by all three Pols in other eukaryotes, despite a lack of TATA elements in their promoters. We are using the yeast system to further our understanding of the basic mechanisms of transcription initiation.

Hamada M, Huang Y, Lowe TM, Maraia RJ. Widespread use of TATA elements in the core promoters

for RNA polymerases III, II, and I in fission yeast. Mol Cell Biol 2001;21:6870-6881.

Huang Y, Hamada M, Patel J, Maraia RJ. Construction of FLAG and histidine tagging vectors for

Schizosaccharomyces pombe. Yeast 2001;18:463-468.

Huang Y, Maraia RJ. Comparison of the RNA polymerase III transcription machinery in S. pombe,

S. cerevisiae and humans (review). Nucleic Acids Res 2001;29:2675-2690.

Huang Y, McGillicuddy E, Weindel M, Dong S, Maraia R. The fission yeast TFIIB-related factor

limits RNA polymerase III to a TATA-dependent pathway of TBP recruitment. Nucleic Acids Res 2003;31:2108-2116.
 

aKristina Treanor, BS, former Postbaccalaureate Fellow

bClaire Vech, BS, MS, left NICHD in 2003

cAmy Mozlin, BS, former Postbaccalaureate Fellow  
dEdward McGillicuddy, BS, former Postbaccalaureate Fellow 

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