The complexity inherent in the production of ribosomal RNAs (rRNAs), the key RNA components of the ribosome, is interesting in itself. The P-A3 intermediate and its direct precursor 35S(P) were both readily detected by the 5′ ETS probe p23 (Fig. 2019 Sep;31(9):1945-1967. doi: 10.1105/tpc.18.00874. 2019 Sep 19;47(16):8649-8661. doi: 10.1093/nar/gkz679. Additional sequences in the 3′ extremities of these clones are marked in red lowercase letters. In the major ITS1-first pathway, the 35SP transcript is split at ITS1 endonucleolytic site A3 into P-A3 and 27SA3 precursors. In the present study, we characterized an Arabidopsis Pumilio-encoding gene, APUM23. 2018 Apr;64(2):393-404. doi: 10.1007/s00294-017-0764-x. The 27SA3 intermediates identified by primers 27P1 and 27P2 were validated by sequencing of 22 independent clones (E). 4, A, B, and D). Fernández-Pevida A, Kressler D, de la Cruz J. Wiley Interdiscip Rev RNA. In a screen for MAS2 interactors, we identified RIBOSOMAL RNA PROCESSING 7 (RRP7), an ortholog of yeast rRNA … Processing of ribosomal RNAs (rRNAs) is an essential step in ribosome biogenesis and begins with transcription of the rDNA. The number of identical clones are indicated to the left (A) and right (B) of each fragment, respectively. The P-A3 intermediates identified by primers 18P6, 18P7, 18P3, and 18P4 were validated by sequencing of 87 independent clones (F). Dysfunction of ribosomal biogenesis (Gallagher et al., 2004; Ferreira-Cerca et al., 2005, 2007; Tafforeau et al., 2013) results in severe developmental defects in higher plants (Byrne, 2009; Fujikura et al., 2009; Horiguchi et al., 2011; Weis et al., 2015a, 2015b) and serious genetic diseases in mammals (Choesmel et al., 2007; Narla and Ebert, 2010; McCann and Baserga, 2013; Sondalle and Baserga, 2014; Bai et al., 2016). The remaining seedlings were transferred to precooled water and treated in a dark growth chamber at 4°C. After amplification for 35 cycles, bands obtained by cRT-PCR were subcloned into the pEasy-T vector (Transgene; CT101-02; Supplemental Fig. Different rRNA precursors are marked. 2 and 3). The number of identical clones is indicated to the right of each fragment. These intermediates were then amplified by pairs of PCR primers, and the resulting amplification products were verified by sequencing (Supplemental Fig. Cannabidiol Cannabidiol (CBD) is a cannabinoid found in cannabis. A and…, Mapping of the 5′ and 3′ extremities of the 35S(P) and 32S transcripts.…, Northern blots to detect pre-rRNA processing in rice. TruSeq Stranded Total RNA with Ribo-Zero Plant | For plant transcriptome studies Capture a comprehensive view of the plant transcriptome with this RNA-Seq library prep workflow. S4). Northern blots to detect pre-rRNA processing in rice. S5D). The number of clones with additional sequences, such as polyadenylation at the 3′ end, is marked in parentheses. Many RBFs are involved in the processing of the primary ribosomal (r)RNA transcript, in which three of the four rRNAs are imbedded. 4D). The 27SA3 intermediates identified by primers 27P1 and 27P2 were validated by sequencing of 22 independent clones (E). Ribosomal RNA genes in plants are highly variable both in copy number and in intergenic spacer (IGS) length. In higher plants, the U3 small nucleolar ribonucleoprotein (U3 snoRNP) was first purified from cauliflower inflorescences as the Nuclear Factor D complex (Sáez-Vasquez et al., 2004a, 2004b) and from Brassica oleracea as BoU3 (B. oleracea U3) complex (Samaha et al., 2010). S11). 1–4; Supplemental Fig. S6A, S7A, and S7B; Supplemental Table S1). 6). For each fragment, the number of clones obtained is indicated on the right. Moreover, the 3′ ends of the 35S(P) precursors were polyadenylated, which was rarely detected in the 32S precursors (Fig. japonica; Huang et al., 2012). The processing sites and rRNA intermediates have been well defined in budding yeast (Saccharomyces cerevisiae), revealing the detailed mechanism of ribosome biogenesis and pre-rRNA processing in eukaryotes (Venema and Tollervey, 1999; Henras et al., 2015). 5C), S7, and p42 (Fig. Additional sequences in the 3′ extremities of these clones are marked in red lowercase letters. 5 and 7; Supplemental Figs. Matured rRNAs stained with MB serve as the loading control. The probes p23, S7, and p42 (Fig. Epub 2019 Jun 25. Cold-sensitive mutants defective in ribosome assembly, Alternative pre-rRNA processing pathways in human cells and their alteration by cycloheximide inhibition of protein synthesis, Circular RT-PCR assay using Arabidopsis samples, Arabidopsis protein arginine methyltransferase 3 is required for ribosome biogenesis by affecting precursor ribosomal RNA processing, An overview of pre-ribosomal RNA processing in eukaryotes, The 5′ end of yeast 5.8S rRNA is generated by exonucleases from an upstream cleavage site, Nascent RNA sequencing reveals distinct features in plant transcription, Differential contributions of ribosomal protein genes to, The DEAD-box RNA helicase AtRH7/PRH75 participates in pre-rRNA processing, plant development and cold tolerance in Arabidopsis, A map of rice genome variation reveals the origin of cultivated rice, The RNA helicase Mtr4p modulates polyadenylation in the TRAMP complex, Structural heterogeneity in pre-40S ribosomes, Cold shock induces a major ribosomal-associated protein that unwinds double-stranded RNA in, Translational dynamics revealed by genome-wide profiling of ribosome footprints in Arabidopsis, Ribosome biogenesis and the translation process in, Inside the 40S ribosome assembly machinery, Differential mRNA translation contributes to gene regulation under non-stress and dehydration stress conditions in, The 5′ external transcribed spacer in mouse ribosomal RNA contains two cleavage sites, Yeast pre-rRNA processing and modification occur cotranscriptionally, Architecture of the 90S pre-ribosome: A structural view on the birth of the eukaryotic ribosome, RT-PCR analysis of 5′ to 3′-end-ligated mRNAs identifies the extremities of cox2 transcripts in pea mitochondria, Arabidopsis AtRRP44A is the functional homolog of Rrp44/Dis3, an exosome component, is essential for viability and is required for RNA processing and degradation, RNA degradation by the exosome is promoted by a nuclear polyadenylation complex, A ‘garbage can’ for ribosomes: how eukaryotes degrade their ribosomes, An RNA conformational switch regulates pre-18S rRNA cleavage, The exosome and 3′-5′ RNA degradation in plants, Degradation of a polyadenylated rRNA maturation by-product involves one of the three RRP6-like proteins in, Polyadenylation-assisted RNA degradation processes in plants, MTR4, a putative RNA helicase and exosome co-factor, is required for proper rRNA biogenesis and development in, The RNA helicases AtMTR4 and HEN2 target specific subsets of nuclear transcripts for degradation by the nuclear exosome in, Regulation of Pol I-transcribed 45S rDNA and Pol III-transcribed 5S rDNA in Arabidopsis, Functional separation of pre-rRNA processing steps revealed by truncation of the U3 small nucleolar ribonucleoprotein component, Mpp10, Molecular signature of chilling adaptation in rice, Natural alleles of a proteasome α2 subunit gene contribute to thermotolerance and adaptation of African rice, Structural basis for site-specific ribose methylation by box C/D RNA protein complexes, Widespread translational control contributes to the regulation of Arabidopsis photomorphogenesis, Translational landscape of photomorphogenic Arabidopsis, A cold-inducible DEAD-box RNA helicase from, Alternative pathways in the processing of ribosomal RNA precursor in, Rice LTG1 is involved in adaptive growth and fitness under low ambient temperature, Origins and activities of the eukaryotic exosome, Structural snapshot of cytoplasmic pre-60S ribosomal particles bound by Nmd3, Lsg1, Tif6 and Reh1, A second base pair interaction between U3 small nucleolar RNA and the 5′-ETS region is required for early cleavage of the yeast pre-ribosomal RNA, Ribosome biogenesis and cell growth: mTOR coordinates transcription by all three classes of nuclear RNA polymerases, Specific contacts between protein S4 and ribosomal RNA are required at multiple stages of ribosome assembly, 40S ribosome biogenesis co-factors are essential for gametophyte and embryo development, The 3′ end of yeast 5.8S rRNA is generated by an exonuclease processing mechanism, At the crossroads of growth control; making ribosomal RNA. 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