Journal: Nucleic Acids Research

Article Title: The conserved ribonuclease aCPSF1 triggers genome-wide transcription termination of Archaea via a 3′-end cleavage mode

doi: 10.1093/nar/gkaa702

Figure Lengend Snippet: The depletion of Mmp-aCPSF1 causes a genome-wide transcription read-through (TRT). ( A, B ) The strand-specific RNA-seq mapping profiles (left) show 3′-end extensions (dot magenta brackets) of MMP1100 ( A ) and MMP1147 ( B ) in ▽ aCPSF1 (red) referenced to that in strain S2 (blue). Numbers on top indicate the genomic sites, and bullets show genes. Northern blot (middle) assayed TRTs using the respective probes (horizontal sticks in A), and 23S rRNA was used as an internal control. 3′RACE (right) assayed transcription termination sites (TTSs) and TRTs. ( C ) Pair-wise comparison of genome-wide Log2 FPKM ratios of transcription units (TUs) and the intergenic regions (IGRs) between strain S2 and ▽ aCPSF1 . The beneath ruler indicates the genomic location. ( D ) A metaplot diagram shows the average reads mapping pattern of TUs and the flanking IGRs in S2 and ▽ aCPSF1 based on normalization of 750 TUs that have an IGR length >100 nt. ( E ) A flowchart depicts TRT identification and TRT index calculation. ( F ) A diagram shows percentages of each TRT type occurred in ▽ aCPSF1 . Bent arrows and horizontal blue lines indicate TSS and transcript lengths in strain S2, respectively, and dot magenta arrows indicate transcripts that occur TRT in ▽ aCPSF1 . *, TU numbers occurring TRTs. ( G ) Boxplots show the FPKM fold changes of Type II TRT in (F) for the upstream TUs that generate TRTs (TU (Up)) and the tandem downstream TUs (TU (Down)), respectively.

Article Snippet: Four RNA fragments containing the termination sites and the flanking sequences of MMP1100, MMP1149 , MMP0901 and MMP1697 were synthesized by Genscript (China).

Techniques: Genome Wide, RNA Sequencing, Northern Blot, Control, Comparison

Journal: Nucleic Acids Research

Article Title: The conserved ribonuclease aCPSF1 triggers genome-wide transcription termination of Archaea via a 3′-end cleavage mode

doi: 10.1093/nar/gkaa702

Figure Lengend Snippet: The terminator motif of M. maripaludis determined by Term-seq, and the ribonuclease activity of Mmp -aCPSF1 on the motif is essential to transcription termination. ( A ) Using WebLogo (v2.8.2) , a logo representation was generated for the sequence motif upstream of 998 primary TTSs that are defined by Term-seq. ( B ) A metaplot diagram shows the average reads of each 20 nt upstream and downstream of the 998 primary TTSs (dot black line) in 22°C-cultured S2 (blue line) and ▽ aCPSF1 (red line). ( C ) The ribonuclease activity of Mmp -aCPSF1 was assayed on three representative uridine-rich RNAs derived from the TTSs (red bases) flanking sequences of MMP1100 , MMP1149 and MMP0901 (upper). A uridine tract-less RNA fragment from MMP1697 was used as a control. A urea sequencing gel displays the enzymatic products (lower). No, C1, and Mu indicate the assays without, and with addition of Mmp -aCPSF1 and the catalytic inactive mutant (H243A/H246A), respectively. Red and pink arrows point to the presumed cleavage products at TTS and other sites downstream uridine-rich sequences. OH, a hydroxyl ladder indicates migrations of RNA products. ( D ) Western blot assays the Mmp -aCPSF1 protein abundances in three batches of 22°C-grown strains S2, ▽ aCPSF1 , Mmp -com( Mmp -C1) (Com(wt)) and Mmp -com( Mmp -C1mu) (Com(mu)). ( E ) Growth curves of the four strains were assayed on three batches of the 22°C-grown cultures, and the averages and standard deviations are shown. ( F ) 3′RACE assays the TRTs in 22°C-grown strains S2, ▽ aCPSF1 , Com(wt) and Com(mu). Blue and magenta arrows indicate the PCR products of normal terminations (TTSs) and TRTs, respectively. M, a DNA ladder indicates migrations of the PCR products.

Article Snippet: Four RNA fragments containing the termination sites and the flanking sequences of MMP1100, MMP1149 , MMP0901 and MMP1697 were synthesized by Genscript (China).

Techniques: Activity Assay, Generated, Sequencing, Cell Culture, Derivative Assay, Control, Mutagenesis, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: MiR-30a-5p Inhibits Epithelial-to-Mesenchymal Transition and Upregulates Expression of Tight Junction Protein Claudin-5 in Human Upper Tract Urothelial Carcinoma Cells

doi: 10.3390/ijms18081826

Figure Lengend Snippet: Expression profiles of miRNAs in human UTUC tumor and non-tumor samples. Three tumor (T1–T3; light blue to navy blue) and three adjacent normal tissues (N1–N3; orange-yellow-green) were subjected to RNA extraction and NGS-miRNA sequencing through the Illumina MiSeq platform. ( A ) Heat-map clustering analysis was conducted to examine the overall miRNA expression profiles among samples. A line scatterplot was used to present miRNA expression profiles; ( B ) Downregulation of miR-30a-5p in UTUC tumor tissues ( n = 22) compared with adjacent normal controls ( n = 14) by RT-qPCR analysis. The asterisk denotes p < 0.001 using an unpaired t -test.

Article Snippet: The small RNA library construction and deep sequencing was carried out at a Biotechnology Company (Welgene, Taipei, Taiwan).

Techniques: Expressing, RNA Extraction, Sequencing, Quantitative RT-PCR