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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 |
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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 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 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, 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 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, Melvin DePamphilis, PhD, Laboratory of
Molecular Growth Regulation, NICHD, Jack Keene, PhD, Scott Tenenbaum, PhD,
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