Journal: bioRxiv

Article Title: A bifunctional snoRNA with separable activities in guiding rRNA 2’-O-methylation and scaffolding gametogenesis effectors

doi: 10.1101/2024.10.03.615557

Figure Lengend Snippet: a , Total and poly(A)+ RNA-seq profiles of the mamRNA and U14 -encoding genomic locus (n=2). The numbers above the scheme indicate the nucleotide length from mamRNA TSS (black arrow). The numbers in square brackets indicate the range of reads. Below are shown the sequences at the borders of the intron with the 5’ splice site (5’SS), branchpoint (BP) and 3’ splice site (3’SS) highlighted in bold. Nucleotides mutated in dedicated strains are shown below in red. b , Semi-quantitative RT-PCR assay of the mamRNA-U14 locus. Left , scheme showing the different strains used, with point mutations labeled with red stars. The primers used are denoted by green arrows. Right , cDNA analysis on agarose gels. act1 + was used as a loading control and no RT reactions (-RT) were loaded in parallel. c , Northern blots showing mamRNA and U14 levels from total RNA samples, in the indicated genetic backgrounds. The position of the different probes used (A, B, C and D) are depicted on top. Ribosomal RNAs served as loading control (lower panels). d and e , Representative images of mamRNA localization in mitotic cells of the indicated genetic backgrounds, expressing either GFP-tagged Mmi1 or Nop56. smFISH probes used were specific of mamRNA 5’ exon and DNA was stained with DAPI. Images are shown as Z-projections. Scale bar, 5 µm. In d , white arrows point to Mmi1 dot- mamRNA colocalization.

Article Snippet: To delineate the species produced from the mamRNA-U14 locus in wild type cells, we conducted direct RNA sequencing (dRNA-seq) using Oxford Nanopore Technology (ONT) ( ).

Techniques: RNA Sequencing, Quantitative RT-PCR, Labeling, Control, Northern Blot, Expressing, Staining

Journal: bioRxiv

Article Title: A bifunctional snoRNA with separable activities in guiding rRNA 2’-O-methylation and scaffolding gametogenesis effectors

doi: 10.1101/2024.10.03.615557

Figure Lengend Snippet: a , dRNA-seq profile of the mamRNA and U14 -encoding genomic locus (n=2). The numbers in square brackets indicate the range of reads. Below is shown a subset of reads using the following down-sampling parameters in IGV: sampling window size (bases) = 230 ( i.e. median read length); numbers of reads per window = 200. b , Multiple sequence alignment (CLUSTALW) of S. pombe snR107 , S. cerevisiae snR190 and human SNORD12/B/C . The C, D’, C’ and D boxes are highlighted in red. The antisense elements (ASE1 and ASE2) complementary to the 25S or 28S rRNAs are highlighted in magenta and orange respectively. Conserved residues are denoted with an asterisk. Nucleotides immediately upstream of the C box and downstream of the D box were excluded for the analysis. c , Northern blots showing the fractions of snR107 and U14 immunoprecipitated with HTP-tagged versions of Fib1, Nop56, Nop58 and Snu13. An untagged strain was used as negative control. IP = Immunoprecipitate; WCE = Whole Cell Extract. d and e , Representative images of mamRNA and snR107 localization as detected by smFISH in cells of the indicated genetic backgrounds. DNA was stained with DAPI. Images are shown as Z-projections. Scale bar, 5 µm.

Article Snippet: To delineate the species produced from the mamRNA-U14 locus in wild type cells, we conducted direct RNA sequencing (dRNA-seq) using Oxford Nanopore Technology (ONT) ( ).

Techniques: Sampling, Sequencing, Northern Blot, Immunoprecipitation, Negative Control, Staining

Journal: bioRxiv

Article Title: A bifunctional snoRNA with separable activities in guiding rRNA 2’-O-methylation and scaffolding gametogenesis effectors

doi: 10.1101/2024.10.03.615557

Figure Lengend Snippet: a , RNA methylation scores (RMS) of the 25S rRNA residues 2480 to 2493 (complementary to snR107 ASE1) as determined by RiboMethSeq in strains of the indicated genotypes (mean (RMS-median all RMS) ±SD; n=3). b , Scheme depicting the snR107 sequence with color-coded motifs mutated in strains of interest (snR107 ASE1mut , snR107 kloopmut , snR107 21ntΔ , snR107 ASE2mut ). The C, D’, C’ and D boxes are labeled in bold and red. c , RNAseH-based cleavage assay. Left , Scheme depicting the 25S rRNA with the positions of Gm 2483 , the RNA/DNA oligonucleotide used for RNAseH cleavage (green) and the Northern probe (red). Below are shown the 25S rRNA products obtained with or without 2’-O-Me. Right , Northern blot showing 25S rRNA in the presence or absence of the RNA/DNA oligonucleotide, from total RNA samples in the indicated genetic backgrounds. Ribosomal RNAs served as loading control. The blue and red arrows denote the 5’ and 3’ cleavage products, respectively. d , Polysome profiles from total cellular extracts obtained on sucrose gradients in strains of the indicated genetic backgrounds. Absorbance at 254 nm was measured for up to 13 min of collection time and is expressed as arbitrary units (A.U). The grey arrows point to half-mers.

Article Snippet: To delineate the species produced from the mamRNA-U14 locus in wild type cells, we conducted direct RNA sequencing (dRNA-seq) using Oxford Nanopore Technology (ONT) ( ).

Techniques: Methylation, Sequencing, Labeling, Cleavage Assay, Northern Blot, Control

Journal: bioRxiv

Article Title: A bifunctional snoRNA with separable activities in guiding rRNA 2’-O-methylation and scaffolding gametogenesis effectors

doi: 10.1101/2024.10.03.615557

Figure Lengend Snippet: a , Scheme depicting the induction of ectopic meiosis by inhibition of the pat1-as allele ( pat1-L95G ) with 3-MB-PP1, which in turn activates the Mmi1 inhibitor Mei2 and hence prevents meiotic mRNA degradation. b , Northern blots showing meiRNA and snR107 levels from total RNA samples isolated at different time points following addition of 3-MB-PP1 in wild type cells. Ribosomal RNAs served as loading control. V = vegetative cells; -N = nitrogen-starved cells. c , d and e , RT-qPCR analyses of mcp5 + and ssm4 + meiotic mRNA levels upon induction of meiosis in cells of the indicated genetic backgrounds (mean±SD; n=3 or 4; normalized to total RNA concentration, relative to wt -N). Note that data for the wild type strain in c were replotted in d and e to ease comparison. Student’s t-test (two-tailed) was used to calculate p-values (relative to wt). NS = not significant. f , Poly(A)+ RNA-seq analyses of wild type and snR107 kloopmut cells upon meiosis induction (n=2). Shown are the median expression profiles of the Mmi1 regulon and the corresponding heatmaps for each individual gene (relative to wt -N; log2 scale). V = vegetative cells; -N = nitrogen-starved cells.

Article Snippet: To delineate the species produced from the mamRNA-U14 locus in wild type cells, we conducted direct RNA sequencing (dRNA-seq) using Oxford Nanopore Technology (ONT) ( ).

Techniques: Inhibition, Northern Blot, Isolation, Control, Quantitative RT-PCR, Concentration Assay, Comparison, Two Tailed Test, RNA Sequencing, Expressing

Journal: RNA Biology

Article Title: Computational methods for RNA modification detection from nanopore direct RNA sequencing data

doi: 10.1080/15476286.2021.1978215

Figure Lengend Snippet: Schematic overview of direct RNA nanopore sequencing and the strategies to detect RNA modifications . (A) Direct RNA sequencing allows the sequencing of native RNA molecules. As the molecule goes through the nanopore, it causes alterations in the ionic current that is going through the nanopore. These disruptions can be converted into their corresponding nucleotide sequences using machine learning algorithms, such as hidden Markov models or recurrent neural networks. (B) Schematic representation of the two major approaches used to detect RNA modifications in nanopore sequencing data: the detection of RNA modifications in the form of alterations of raw signal intensities (upper panel), or systematic base-calling ‘errors’ (lower panel)

Article Snippet: A promising alternative to sequencing-by-synthesis-technologies is the direct RNA nanopore sequencing (dRNA-seq) platform offered by Oxford Nanopore Technologies (ONT) ( ).

Techniques: Nanopore Sequencing, RNA Sequencing, Sequencing

Journal: RNA Biology

Article Title: Computational methods for RNA modification detection from nanopore direct RNA sequencing data

doi: 10.1080/15476286.2021.1978215

Figure Lengend Snippet: Chronological overview of the community efforts to develop tools to detect and quantify RNA modifications using direct RNA nanopore sequencing

Article Snippet: A promising alternative to sequencing-by-synthesis-technologies is the direct RNA nanopore sequencing (dRNA-seq) platform offered by Oxford Nanopore Technologies (ONT) ( ).

Techniques: Nanopore Sequencing