We study mechanisms of eukaryotic RNA biogenesis and functions of the human La antigen, for which transcription by RNA polymerase III (Pol III) serves as a model system of gene expression. Transfer RNAs are the Pol III transcripts currently under most intense study. 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 ribosomes. Our interests focus on a few of the individual steps in RNA production (e.g., transcription initiation and termination as well as RNA processing and intranuclear trafficking), including coordination of the sequential steps in time and space within the cell. In particular, we have focused on 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 protein that also binds to the 5' untranslated regions of certain mRNAs, regulating and coordinating key steps in RNA expression. We use modern genetics, molecular biology, and biochemical approaches and rely heavily on analytical biochemistry as well as tissue culture, yeast systems, and transgenic and gene-altered mice.

Functions of the Human La Antigen in RNA Expression
Maraia, Intine, Schwartz, Mukhergee, Muse, Nelson, Treanor
Human La antigen is a nuclear phosphoprotein that is present in all eukaryotic cells thus far examined. 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, which remains associated with newly synthesized transcripts to direct their maturation. As such, La is considered a regulatory chaperone for nascent RNAs because it can control their nuclear residence and accessibility to the processing enzymes. In part, La functions as a regulatory chaperone 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 to interact with the initiating pppG of the nascent transcript and to activate transcription initiation by Pol III, implying that these activities are mechanistically related. The results suggest an “La cycle” of transcription and post-transcriptional regulation that is mediated in part by the C-terminal domain (CTD) of La. Others have recently shown that La is dephosphorylated at S366 during early apoptosis. We have developed two sets of monospecific antibodies that can distinguish phosphoS366 (pLa) from nonphospohoS366 (npLa). We could demonstrate that npLa and pLa exhibit distinct subnuclear localizations (nucleoplasm versus nucleoli) and that they are differentially associated with certain RNAs in vivo.

Our data point to 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 tRNA processing activities. In this case, La recognizes and normally binds to its substrate pre-tRNAs, but the defect causes improper routing in the nucleus, resulting in the accumulation of a dead-end tRNA processing intermediate that is nonfunctional. Our studies revealed that La traffics through the nucleus and is exported to the cytoplasm, and have uncovered an unexpected nuclear export signal (NES) in La. The existence of an efficient, carrier-mediated NES system for La is surprising because the established functions of La in Pol III transcript biogenesis are entirely intranuclear. Further characterization revealed that both the nuclear export pathway used for La and other features of La’s 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 include examination of the involvement of La in the expression of certain target mRNAs, especially those whose expression is critical to growth and development.

Transcription Termination by RNA Polymerase III
Maraia, Huang, McGillicuddy, Mozlin
As indicated above, La protein binds to the 3' terminal motif of nascent transcripts, UUU-OH, that 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); the enzyme’s activity can be monitored by using an in vivo colorimetric plate assay. We demonstrated that the expression of the gene in S. pombe depends 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 the toxin a-amanitin and the sensitivity 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 shown that the tRNA gene under study 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 is attributable to a defect in transcription rate, nascent RNA processing, or both; and the nature of other factors that contribute to the La-dependent activation of this tRNA gene.

Transcription Initiation by RNA Polymerase III
Maraia, Huang, Mozlin
Though long interested in transcriptional termination, we have made some recent discoveries that 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, which both function at the core promoter. Homologs of these factors exist in all eukaryotes and archaea and play a central role 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 signals from distal regulatory elements. Genome-wide 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 extended the results to large rRNA synthesis, as mutation of the TATA element in the Pol I promoter also abolished rRNA expression in fission yeast. The results point to TATA-unified transcription systems in contemporary eukaryotes and provide insight into the residual need for TBP by all three RNA polymerases in other eukaryotes despite a lack of TATA elements in their promoters.