Journal: Nucleic Acids Research

Article Title: Pol5 is required for recycling of small subunit biogenesis factors and for formation of the peptide exit tunnel of the large ribosomal subunit

doi: 10.1093/nar/gkz1079

Figure Lengend Snippet: Depletion of Pol5 impairs pre-rRNA processing at the A2 and C2 cleavage sites. ( A ) Schematic representation of pre-rRNA processing in S. cerevisiae . Positions of the probes used for northern blot analysis and primer extension are indicated and named according to the region where they hybridize. ( B ) Northern blot analysis of RNAs produced during a metabolic labeling experiment. Cells expressing Pol5 from a plasmid (Pol5) or containing an empty plasmid backbone (Depletion) were cultivated in glucose-containing medium for the indicated times (hours) to allow depletion of the endogenously expressed Pol5. At the indicated times, cells were treated for 20 minutes with 4tU. Total RNA was extracted, resolved by gel electrophoresis in denaturing agarose (upper panels) or acrylamide gels (lower panels) and transferred to a nylon membrane. To detect labeled RNAs, membranes were processed as indicated in methods. NL indicates RNAs derived from cells not treated with 4tU. ( C ) Northern blot analysis of RNAs with radioactively labeled probes. Total RNA obtained at indicated depletion times (in hours) was resolved in denaturing agarose (top four panels) or acrylamide (bottom three panels) gels and transferred to a nylon membrane, which was hybridized with the indicated oligonucleotide probes. ( D ) Primer extension analysis of RNAs obtained during Pol5 depletion. Total RNAs obtained at the indicated depletion times (hours) were used in primer extension assay with the indicated oligonucleotides. Reaction products were separated by denaturing polyacrylamide gel electrophoresis and radioactively labeled cDNA fragments were detected. Sequencing reactions on a plasmid (K375) encoding a full ribosomal DNA copy were performed in parallel (lanes G, A, T and C).

Article Snippet: His-TEV protease cleavage site-ProteinA (HTP)-tagged Pol5 was crosslinked to associated RNAs in culturo using UV light at 254 nm.

Techniques: Northern Blot, Produced, Labeling, Expressing, Plasmid Preparation, Nucleic Acid Electrophoresis, Membrane, Derivative Assay, Primer Extension Assay, Polyacrylamide Gel Electrophoresis, Sequencing

Journal: Nucleic Acids Research

Article Title: Pol5 is required for recycling of small subunit biogenesis factors and for formation of the peptide exit tunnel of the large ribosomal subunit

doi: 10.1093/nar/gkz1079

Figure Lengend Snippet: Pol5 crosslinks to the 5′ ETS, ITS2 and domain III of the 25S rRNA sequence. ( A ) Wild-type yeast or yeast cells expressing C-terminally HTP tagged Pol5 were crosslinked in culturo using UV light. RNA-protein complexes were affinity purified, and RNAs were trimmed, 5′ labeled using 32 P and ligated to adaptors. Complexes were separated by denaturing polyacrylamide gel electrophoresis, transferred to a nylon membrane and visualized by autoradiography. ( B ) RNAs isolated from the membrane shown in (A) were reverse transcribed, PCR amplified and subjected to next-generation sequencing. The obtained sequence reads were mapped to the S. cerevisiae genome and the relative proportions of reads mapping to genes encoding different types of RNAs is shown. rRNA – ribosomal RNA, tRNA – transfer RNA, snoRNA – small nucleolar RNA, snRNA – small nuclear RNA, ncRNA – non-coding RNA, IR – intergenic region, mRNA – messenger RNA. ( C ) The normalized numbers of reads mapping to each nucleotide of the RDN37 locus encoding the 35S pre-rRNA is shown above a schematic view of the transcript. ( D ) The number of sequencing reads in the Pol5-HTP dataset mapping to each nucleotide of the 25S rRNA sequence is shown on the secondary structure of the mature 25S rRNA using a color code in which the maximum number of reads is shown in red and lower numbers of reads are shown in yellow. ( E ) The number of sequencing reads in the Pol5-HTP dataset mapping to each nucleotide of the 5′ ETS sequence was mapped onto the tertiary structure of the SSU processome purified via Utp1 and Kre33 (PDB: 5TZS) using a color code as in (D). The t-UTP proteins are highlighted in different colors and labeled. The rRNA sequences and the U3 snoRNA are shown as ribbons in grey and blue respectively, and overlay the RPs and assembly factors, which are shown in pale cyan.

Article Snippet: His-TEV protease cleavage site-ProteinA (HTP)-tagged Pol5 was crosslinked to associated RNAs in culturo using UV light at 254 nm.

Techniques: Sequencing, Expressing, Affinity Purification, Labeling, Polyacrylamide Gel Electrophoresis, Membrane, Autoradiography, Isolation, Reverse Transcription, Amplification, Next-Generation Sequencing, Purification

Journal: Nucleic Acids Research

Article Title: Pol5 is required for recycling of small subunit biogenesis factors and for formation of the peptide exit tunnel of the large ribosomal subunit

doi: 10.1093/nar/gkz1079

Figure Lengend Snippet: Association of Pol5 and other AFs with pre-rRNAs. ( A ) Pre-ribosomal complexes were isolated via TAP-tagged assembly factors. Whole cell lysates (WCL) and affinity purification samples (IP) were analyzed by western blotting with the indicated antibodies. TAP-tagged proteins are indicated and C indicates the non-tag-containing strain (BY4741) used as a control. ( B , C ) Northern blot analyses of pre-rRNAs co-purified with AFs. RNAs enriched in the affinity purifications were extracted and resolved in denaturing acrylamide (B) or agarose (C) gels, transferred to nylon membrane, and hybridized with the indicated, radiolabeled oligonucleotide probes.

Article Snippet: His-TEV protease cleavage site-ProteinA (HTP)-tagged Pol5 was crosslinked to associated RNAs in culturo using UV light at 254 nm.

Techniques: Isolation, Affinity Purification, Western Blot, Control, Northern Blot, Purification, Membrane

Journal: Nucleic Acids Research

Article Title: Pol5 is required for recycling of small subunit biogenesis factors and for formation of the peptide exit tunnel of the large ribosomal subunit

doi: 10.1093/nar/gkz1079

Figure Lengend Snippet: Depletion of Pol5 affects the recruitment of AFs and RPs that bind pre-60S complexes around the peptide exit tunnel. ( A ) Heatmap of LSU ribosomal proteins identified in the qMS analysis of Noc2- and Rlp7-associated particles. Only proteins identified in two independent experiments are shown (proteins not observed in the individual experiment are depicted in gray). iTRAQ ratios (Pol5 expression versus Pol5 depletion) were calculated to determine the relative abundance of proteins in the two samples. Color code indicates the log2 of iTRAQ ratios. ( B ) Structure of a pre-60S complex purified via Ytm1 (PDB: 6ELZ) with the binding site of Pol5 and RPs affected by Pol5 depletion highlighted. The rRNA is shown in cartoon view in dark gray and the crosslinking sites of Pol5 and Sbp4 are marked in red-yellow and purple respectively. RP and AFs are depicted in surface view, generally in pale cyan with the exception of ribosomal proteins depleted in pre-60S particles isolated from cells lacking Pol5, which are shown in colors and labeled. ( C ) Heatmap of AFs identified in the qMS analysis of Noc2- and Rlp7-associated particles. Color code as in A. ( D ) Structure of a pre-60S particle purified via Nog2 (PDB: 3JCT) with the binding sites of Pol5, Spb4 and selected AFs highlighted. Presentation styles and colors are as in (B) Domains IV and VI of 25S are indicated and separated by a dashed line.

Article Snippet: His-TEV protease cleavage site-ProteinA (HTP)-tagged Pol5 was crosslinked to associated RNAs in culturo using UV light at 254 nm.

Techniques: Multiplex sample analysis, Expressing, Purification, Binding Assay, Isolation, Labeling

Journal: Nucleic Acids Research

Article Title: Pol5 is required for recycling of small subunit biogenesis factors and for formation of the peptide exit tunnel of the large ribosomal subunit

doi: 10.1093/nar/gkz1079

Figure Lengend Snippet: Depletion of Pol5 impairs the association of Spb4 and Nop53 with pre-rRNAs. ( A ) Pre-60S particles from different stages of 60S maturation were purified via TAP-tagged AFs either in the absence or presence of Pol5 (D and P respectively). Whole cell lysates (WCL) and affinity purification samples (IP) were analyzed by western blotting with the indicated antibodies. TAP-tagged proteins are indicated, C indicates the non-tagged strain BY4741 used as a control. ( B ) Northern blot analysis of pre-rRNAs co-purified with different AFs. RNAs enriched in the affinity purifications were extracted and resolved on denaturing agarose or acrylamide gels, transferred to nylon membranes, and hybridized with the indicated oligonucleotide probes.

Article Snippet: His-TEV protease cleavage site-ProteinA (HTP)-tagged Pol5 was crosslinked to associated RNAs in culturo using UV light at 254 nm.

Techniques: Purification, Affinity Purification, Western Blot, Control, Northern Blot

Journal: Nucleic Acids Research

Article Title: Pol5 is required for recycling of small subunit biogenesis factors and for formation of the peptide exit tunnel of the large ribosomal subunit

doi: 10.1093/nar/gkz1079

Figure Lengend Snippet: Depletion of Pol5 affects the release of t-Utp proteins from 5′ ETS-containing pre-rRNAs. ( A ) Schematic representation of alternative pre-rRNA processing pathways for removal of the 5′ ETS sequences of the 35S pre-rRNA. Positions of the primers used for northern blot analysis are indicated and named by the region where they hybridized. ( B ) Complexes associated with TAP-tagged t-Utps were purified in the absence or presence of Pol5 (D and P respectively). Whole cell lysates (WCL) and affinity purification samples (IP) were analyzed by western blot with indicated antibodies. TAP-tagged proteins are indicated, C indicates the non-tagged strain BY4741 used as a control. ( C , D ) Northern blot analysis of RNAs co-purified with t-Utps. RNAs enriched in the affinity purifications were extracted and resolved on denaturing agarose (C) or acrylamide (D) gels, transferred to nylon membrane, and hybridized with the indicated oligonucleotide probes.

Article Snippet: His-TEV protease cleavage site-ProteinA (HTP)-tagged Pol5 was crosslinked to associated RNAs in culturo using UV light at 254 nm.

Techniques: Northern Blot, Purification, Affinity Purification, Western Blot, Control, Membrane