mtrna sequences  (New England Biolabs)

Journal: Nucleic Acids Research

Article Title: Trypanosome RNA helicase KREH2 differentially controls non-canonical editing and putative repressive structure via a novel proposed ‘bifunctional’ gRNA in mRNA A6

doi: 10.1093/nar/gkad453

Figure Lengend Snippet: KREH2 knockdown effect on a panel of editing mRNAs in PCF and BSF cells. ( A ) Growth curve of KREH2-RNAi ± Tet in PCF and BSF cells. ( B ) Western blot of PCF and BSF mitochondrial extracts with KREH2-RNAi ± Tet at the indicated days post-induction. KREH2 and RESC1/2 were examined. RESC1/2 were used to control for loading and secondary effects, e.g. stability of other extract proteins. ( C ) Heat map of RT-qPCR assays of a panel of fully edited and pre-edited transcripts upon KREH2-RNAi in PCF (left) and BSF (right) total mtRNA. Plotted values represent the log10 relative abundance of –Tet vesus +Tet samples. Assays were normalized to a housekeeping gene (TERT) and the –Tet control. Plotted values are the average of three biological replicates per condition ( n = 3). Mitochondrial ND4 mRNA and 12S and 9S rRNA transcripts do not undergo editing. ( D ) Immunofluorescent microscopy of BSF T. brucei cells. Cells were imaged for marker proteins in editing complexes: KH2F1 (REH2C), RESC1/2 (RESC) or KREL1 (RECC). White arrows point to DAPI-stained kDNA in each cell.

Article Snippet: We also found equivalent isoforms in Illumina-sequenced RESC-bound and total mtRNA transcripts of the PCF strain Lister 427 used here (transcript 4) , and in total mtRNA of strain EATRO 164, including gCR4(186–228) (transcripts 5–7) ( ).

Techniques: Western Blot, Quantitative RT-PCR, Microscopy, Marker, Staining

Journal: Nucleic Acids Research

Article Title: Trypanosome RNA helicase KREH2 differentially controls non-canonical editing and putative repressive structure via a novel proposed ‘bifunctional’ gRNA in mRNA A6

doi: 10.1093/nar/gkad453

Figure Lengend Snippet: Analyses of A6 NC/C ratios and effect of KREH2-RNAi or KH2F1-RNAi on mtRNA or RESC. ( A ) Site-by-site NC/C ratios in PCF versus BSF mtRNA (Mito) across gRNA blocks 1–2. NC/C ratios are scored as the percentage of non-canonical reads divided by the percentage of canonical reads at the same site. Sites 31–38 (highlighted) exhibited particularly high NC/C ratios. –Tet replicates were used ( n = 3). ( B ) Cumulative NC/C ratios in PCF versus BSF mtRNA ± KREH2-RNAi. Note the opposite effect of KREH2-RNAi on editing accuracy between the two stages, i.e. editing accuracy decreased in PCF but increased in BSF cells, within the sites examined. ( C ) Cumulative NC/C ratio in PCF versus BSF mtRNA ± KREH2-RNAi. Ratios are through site 70. ( D ) Same as (C) but in PCF mtRNA ± KH2F1-RNAi. ( E ) Site-by-site and ( F ) cumulative NC/C ratio in PCF RESC ± KREH2-RNAi across gRNA blocks 1–2. KREH2-RNAi-enhanced major intrinsic editing pause sites (MPSs) are annotated (diamonds). ( G ) Cumulative NC/C ratio in PCF RESC ± KREH2-RNAi through site 70. ( H ) The same as (G) but in PCF RESC ± KH2F1-RNAi. Full-amplicon analyses in this study, including the number of replicates, and P -values are also included ( ; ). ( I ) KREH2-RNAi enhanced MPSs. Dotted lines score the fold increase in NC/C ratio upon KREH2-RNAi at the site just 5′ of each intrinsic MPS (diamonds). When comparing two sets of samples (conditions), one set included at least three biological replicates and the other at least two, enabling P -value, average and SD calculations. ±Tet also labeled as ±T.

Article Snippet: We also found equivalent isoforms in Illumina-sequenced RESC-bound and total mtRNA transcripts of the PCF strain Lister 427 used here (transcript 4) , and in total mtRNA of strain EATRO 164, including gCR4(186–228) (transcripts 5–7) ( ).

Techniques: Amplification, Labeling

Journal: Nucleic Acids Research

Article Title: Trypanosome RNA helicase KREH2 differentially controls non-canonical editing and putative repressive structure via a novel proposed ‘bifunctional’ gRNA in mRNA A6

doi: 10.1093/nar/gkad453

Figure Lengend Snippet: An abundant non-canonical 3′-HFE sequence in A6 in PCF and BSF cells, and its modulation by helicase complex REH2C proteins. ( A ) Canonically edited A6 3′ terminus. Color-coded letters are just 3′ of sites for sites requiring: insertion (red), deletion (blue) or changes (black). The ORF, 3′-UTR and never-edited regions are indicated. The first editing site (ES1) is at position 25 counting from the 3′ end. Illumina sequenced gRNA isoforms: gRNA-1 (gA6-1 B1.alt ) in strain LISTER 427 and gRNA-1 gA6 (774–822) in strain EATRO 164 exhibit identical guiding function at block 1, and predicted gRNA-2 m0_306(II)_gA6_v2 [724–766] in strain EATRO 1125 at block 2, produced the best match with canonically edited A6 ( , ). Color-coded arrowheads indicate sites that contain a dominant NC read representing >30% (black) of all reads in RESC + Tet (see below) or lower (see the color scale). ( B ) Actual percentage of the dominant NC read at each indicated site versus all reads (black) or versus all NC reads (white) in PCF mtRNA (Mito) or RESC, and BSF mtRNA ± KREH2-RNAi. The indicated range 38–31 includes only sites with dominant NC reads (>30% or >20%) in (A). ( C ) The 3′-HFE made by the dominant NC reads at sites 31–38. The top two 3′-HFE isoforms found in all PCF and BSF samples examined show the dominant NC reads in gray. General 3′-HFE long or short forms, where ‘n’ represents any T number at sites 35 and 36. Bottom: ∼42 nt extended 3' element, including the 3′-HFE and 3′-terminal pre-edited sequences. ( D ) Frequency of 3′-HFEs in PCF mtRNA or RESC, and BSF mtRNA in the indicated KREH2 or KH2F1 knockdowns. ( E ) Frequency of canonically edited block-1 in PCF mtRNA or RESC and BSF mtRNA ± KREH2-RNAi. ( F ) Frequency of 3′-HFEs, canonically edited block-1, pre-edited and other NC (‘partial’) reads in PCF and BSF ± KREH2-RNAi. When comparing two sets of samples (conditions), one set included at least three biological replicates and the other at least two, enabling P -value, average and SD calculations. ±Tet also labeled ±T.

Article Snippet: We also found equivalent isoforms in Illumina-sequenced RESC-bound and total mtRNA transcripts of the PCF strain Lister 427 used here (transcript 4) , and in total mtRNA of strain EATRO 164, including gCR4(186–228) (transcripts 5–7) ( ).

Techniques: Sequencing, Blocking Assay, Produced, Labeling

Journal: Nucleic Acids Research

Article Title: Trypanosome RNA helicase KREH2 differentially controls non-canonical editing and putative repressive structure via a novel proposed ‘bifunctional’ gRNA in mRNA A6

doi: 10.1093/nar/gkad453

Figure Lengend Snippet: Experimentally determined structure of 3′-HFE-containing full-length A6 and analyses of mRNA RESC association. DMS-MaPseq secondary structure of in vitro transcribed full-length A6: ( A ) pre-edited (403 nt) and ( B ) one of the top two 3′-HFE-containing A6 isoforms (426 nt) in all samples. Diagrams depict 300 nt on the 3′ end of each construct. Sites 31–38 with non-canonical edits in the 3′-HFE and/or sites matching guiding and anchor regions of anti-initiator gRNA (black line) and canonical initiator gRNA-1 (blue line) are annotated. Nucleotides are colored by DMS reactivity calculated as the average ratiometric signal per position across two biological replicates normalized to the highest number of reads in the displayed region, which is set to 1.0. ( C ) Relative DMS reactivity at each nucleotide normalized to the signal in the pre-edited molecule for the same nucleotide. The dotted black box indicates sites 31–38 which had the greatest loss in DMS reactivity for any region in the 3′-HFE-bearing molecule.

Article Snippet: We also found equivalent isoforms in Illumina-sequenced RESC-bound and total mtRNA transcripts of the PCF strain Lister 427 used here (transcript 4) , and in total mtRNA of strain EATRO 164, including gCR4(186–228) (transcripts 5–7) ( ).

Techniques: In Vitro, Construct

Journal: Nucleic Acids Research

Article Title: Trypanosome RNA helicase KREH2 differentially controls non-canonical editing and putative repressive structure via a novel proposed ‘bifunctional’ gRNA in mRNA A6

doi: 10.1093/nar/gkad453

Figure Lengend Snippet: Model of REH2C-dependent developmental control of non-canonical editing by a putative bifunctional gRNA in A6. Amidst extensive RNA editing of unclear function, KREH2, a REH2C-associated helicase, controls programmed (i.e. gRNA-directed and regulated) non-canonical editing. A novel putative bifunctional gCR4 gRNA directs non-canonical editing to generate a 3′ element in A6 besides serving canonically in CR4 editing. This novel gRNA-directed function on A6 seems most active in RESC and is modulated differently by KREH2 during the life cycle. In the PCF, REH2C negatively controls gCR4 gRNA action on non-cognate A6. Loss-of-function mutants (KREH2 or KH2F1 knockdowns) up-regulate the 3′ element to an astonishing ∼35% of RESC-bound A6 molecules. REH2C may positively control gCR4 gRNA action on its cognate target, as is generally expected for canonical editing. The 3′ element forms a stable structure that sequesters the 3′-UTR and blocks canonical editing initiation-mediated ‘repair’ of the 3′-UTR, representing a new type of attenuation. In BSF cells, this element is particularly abundant (∼40%) but not up-regulated by KREH2-RNAi. Instead, KREH2 may be required to maintain the 3′ element at high levels in BSF cells. However, A6 maturation occurs in both stages, but apparently at different levels. Thus, KREH2 differentially controls a novel potentially regulatory gRNA, a putative repressor, preventing excessive A6 maturation in mitochondria. Overall, the current study identified the first example of programmed non-canonical editing, which is both mitochondrial genome encoded and regulated and may control mitochondrial physiology during development. Our model suggests that at least some mitochondrial non-canonical editing became fixed and regulated in the long evolutionary history of kinetoplastids.

Article Snippet: We also found equivalent isoforms in Illumina-sequenced RESC-bound and total mtRNA transcripts of the PCF strain Lister 427 used here (transcript 4) , and in total mtRNA of strain EATRO 164, including gCR4(186–228) (transcripts 5–7) ( ).

Techniques: