Abstract
Although parvulin (Par14/eukaryotic parvulin homolog), a peptidyl-prolyl cis-trans isomerase, is found associated with the preribosomal ribonucleoprotein (pre-rRNP) complexes, its roles in ribosome biogenesis remain undetermined. In this study, we describe a comprehensive proteomics analysis of the Par14-associated pre-rRNP complexes using LC-MS/MS and a knockdown analysis of Par14. Together with our previous results, we finally identified 115 protein components of the complexes, including 39 ribosomal proteins and 54 potential trans-acting factors whose yeast homologs are found in the pre-rRNP complexes formed at various stages of ribosome biogenesis. We give evidence that, although Par14 exists in both the phosphorylated and unphosphorylated forms in the cell, only the latter form is associated with the pre-40 S and pre-60 S ribosomal complexes. We also show that Par14 co-localizes with the nucleolar protein B23 during the interphase and in the spindle apparatus during mitosis and that actinomycin D treatment results in the exclusion of Par14 from the nucleolus. Finally we demonstrate that knockdown of Par14 mRNA decelerates the processing of pre-rRNA to 18 and 28 S rRNAs. We propose that Par14 is a component of the pre-rRNA complexes and functions as an rRNA processing factor in ribosome biogenesis. As the amino acid sequence of Par14 including that in the amino-terminal pre-rRNP binding region is conserved only in metazoan homologs, we suggest that its roles in ribosome biogenesis have evolved in the metazoan lineage.
Highlights
Parvulin (Par14/eukaryotic parvulin homolog), a peptidyl-prolyl cis-trans isomerase, is found associated with the preribosomal ribonucleoprotein complexes, its roles in ribosome biogenesis remain undetermined
We previously reported that Par14 associates with the preribosomal ribonucleoprotein complexes as well as with many proteins that are implicated in the regulation of microtubule assembly or nucleolar reformation during mitosis [12, 13]
We identified 52 proteins involved in ribosome biogenesis, including ribosomal proteins (RPs) and possible trans-acting factors, primarily using the peptide mass fingerprinting (PMF) method with MALDI-TOF/MS after in-gel protease digestion of individual bands excised from SDS-PAGE gels [12]
Summary
Materials—Mouse fibroblast cell line L929, human embryonic kidney cell line 293EBNA, Lipofectamine, Lipofectamine 2000, OptiMEM medium, and SuperScriptTM were obtained from Invitrogen. The membranes were incubated either with the primary anti-Par serum or affinity-purified antibodies in PBS containing 5% nonfat milk and 0.1% (w/v) Tween 20; washed three times for 5 min with PBS, 0.1% Tween 20; and detected with alkaline phosphatase-conjugated secondary antibodies using the nitro blue tetrazolium/5-bromo-4chloro-3-indolyl phosphate stock solution according to the manufacturer’s instructions (Roche Diagnostics). The same transferred membrane was stained with methylene blue for visualizing 28 and 18 S ribosomal RNAs. For metabolic labeling of RNA with L-[methyl-3H]methionine, subconfluent siRNA-transfected 293EBNA cells were incubated for 30 min in medium containing L-[methyl-3H]methionine (50 Ci/ml; GE Healthcare) after 30-min preincubation in methionine-free medium. The cells were chased in medium containing a 10-fold excess of nonradioactive methionine after which RNA was isolated using RNAgent, and 5 g of total RNA was analyzed as described above
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