During ribosome biogenesis, ribosomal RNAs acquire various chemical modifications that ensure the fidelity of translation, and dysregulation of the modification processes can cause proteome changes as observed in cancer and inherited human disorders. Here, we report the complete chemical modifications of all RNAs of the human 80S ribosome as determined with quantitative mass spectrometry. We assigned 228 sites with 14 different post-transcriptional modifications, most of which are located in functional regions of the ribosome. All modifications detected are typical of eukaryotic ribosomal RNAs, and no human-specific modifications were observed, in contrast to a recently reported cryo-electron microscopy analysis. While human ribosomal RNAs appeared to have little polymorphism regarding the post-transcriptional modifications, we found that pseudouridylation at two specific sites in 28S ribosomal RNA are significantly reduced in ribosomes of patients with familial dyskeratosis congenita, a genetic disease caused by a point mutation in the pseudouridine synthase gene DKC1. The landscape of the entire epitranscriptomic ribosomal RNA modifications provides a firm basis for understanding ribosome function and dysfunction associated with human disease.

Plant response to drought and hyperosmosis is mediated by the phytohormone abscisic acid (ABA), a sesquiterpene compound widely distributed in various embryophyte groups. Exogenous ABA as well as hyperosmosis activates the sucrose nonfermenting 1 (SNF1)-related protein kinase2 (SnRK2), which plays a central role in cellular responses against drought and dehydration, although the details of the activation mechanism are not understood. Analysis of a mutant of the moss Physcomitrella patens with reduced ABA sensitivity and reduced hyperosmosis tolerance revealed that a protein kinase designated "ARK" (for "ABA and abiotic stress-responsive Raf-like kinase") plays an essential role in the activation of SnRK2. ARK encoded by a single gene in P. patens belongs to the family of group B3 Raflike MAP kinase kinase kinases (B3-MAPKKKs) mediating ethylene, disease resistance, and salt and sugar responses in angiosperms. Our findings indicate that ARK, as a novel regulatory component integrating ABA and hyperosmosis signals, represents the ancestral B3-MAPKKKs, which multiplied, diversified, and came to have specific functions in angiosperms.abscisic acid | Physcomitrella patens | Raf-like kinase | SnRK2

In cytoplasm, the survival of motor neuron (SMN) complex delivers pre-small nuclear RNAs (pre-snRNAs) to the heptameric Sm ring for the assembly of the ring complex on pre-snRNAs at the conserved Sm site [A(U) 4-6 G]. Gemin5, a WD40 protein component of the SMN complex, is responsible for recognizing pre-snRNAs. In addition, Gemin5 has been reported to specifically bind to the m 7 G cap. In this study, we show that the WD40 domain of Gemin5 is both necessary and sufficient for binding the Sm site of pre-snRNAs by isothermal titration calorimetry (ITC) and mutagenesis assays. We further determined the crystal structures of the WD40 domain of Gemin5 in complex with the Sm site or m 7 G cap of pre-snRNA, which reveal that the WD40 domain of Gemin5 recognizes the Sm site and m 7 G cap of pre-snRNAs via two distinct binding sites by respective base-specific interactions. In addition, we also uncovered a novel role of Gemin5 in escorting the truncated forms of U1 pre-snRNAs for proper disposal. Overall, the elucidated Gemin5 structures will contribute to a better understanding of Gemin5 in small nuclear ribonucleic protein (snRNP) biogenesis as well as, potentially, other cellular activities.

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.

Extracellular vesicle (EV) microRNAs are of major interest as potential diagnostic biomarkers in all cancer types. This study aims to identify miRNA profiles of shed microvesicles (sMVs) and exosomes (Exos) secreted from the isogenic colorectal cancer (CRC) cell lines SW480 and SW620 and evaluate their ability to predict CRC. Deep sequencing of miRNAs in parental cell lysates (CLs) and highly-purified sMVs and Exos was performed. We focused on miRNAs enriched in EVs and dysregulated miRNAs in metastatic cells (SW620) relative to primary cancer cells (SW480). We investigated the ability of EV miRNA signatures to predict CRC tumours using 594 tumours (representing different pathological stages) and 11 normal samples obtained from TCGA. In SW480 and SW620 cells we identified 345 miRNAs, of which 61 and 73 were upregulated and downregulated in SW620-CLs compared to SW480-CLs, respectively. Selective distribution of cellular miRNAs into EVs results in distinct miRNA signatures for sMVs and Exos in each cell line. Cross cell line comparisons of EV miRNA profiles reveal a subset of miRNAs critical in CRC progression from primary carcinoma to metastasis. Many miRNAs non-detectable (<5 TPM) in CLs were significantly enriched (>1000 TPM) in secreted EVs. Strikingly, miR-7641 which is non-detectable in SW480-CL but upregulated in SW620-CL is highly enriched in EVs secreted from both cell lines. Pearson correlation analysis demonstrated that EV miRNA profiles can be used to predict CRC tumours with ~96% accuracy. Our findings suggest that EV miRNA profiles from CRC cell lines may allow prediction of CRC tumours, and that miR-7641 may serve as an attractive candidate for the specific, non-invasive diagnosis and prognosis of CRC.