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Journal: Nucleic Acids Research
Article Title: Deployment of non-canonical splicing in tunicate genomes is mediated by divergent U2AF function and changing m6A modification in U1 and U6 snRNA
doi: 10.1093/nar/gkag659
Figure Lengend Snippet: Divergent m6A patterns in larvacean snRNA. ( A ) From left to right, U2-type snRNA identified in maturing adults of F. borealis and O. dioica, C. intestinalis larvae, and HEK293T cells; graph shows the relative signal intensity measured for each snRNA. ( B ) Detection of m6A on snRNA. Bands were identified based on northern blots; graph shows the distribution of m6A signal; and last lane shows results from mouse ES cells. ( C ) Conservation of m6A regulatory genes. Dots represent gene copy number, full border indicates evidence from transcripts and genomic DNA, and dashed border indicates evidence from genomic DNA only. Empty dots represent gene losses. ( D ) Mapping modified adenines at the 5′ end of U1 snRNA and in the ACAGAGA box of U6 snRNA. Schematics represent RT elongation of a radiolabeled primer annealed to either U1 or U6 snRNA (left and right side, respectively). On U1 snRNA, the 5′ terminal m6A induces RT stop in the presence of thymidine analog 4SedTTP. On U6 snRNA, 4SedTTP provokes RT stop at 2′-O-methylated adenines A 47 and A 48 , and pauses provoked at m6A 43 are weaker. Gel images shows RT elongation products. For U1 snRNA, we quantified the intensity of m6A-associated pause by measuring the ratio of cDNA stopped at position U 2 of U1 snRNA relative to full-length cDNA. Bottom values show ratios measured with 4SedTTP minus ratios measured with dTTP.
Article Snippet: After 2 min at 90 °C, the hybridization mix was placed for 2 min at 50 °C, and 10 U Superscript IV reverse transcriptase (Invitrogen) was added in RT (reverse transcription) buffer (50 mM Tris–HCl, 4 mM MgCl 2 , 10 mM DTT, 50 mM KCl, 30 μM dATP, 30 μM dGTP, 30 μM dCTP, pH 8.3) in the presence of 30 μM of either
Techniques: Northern Blot, Modification, Methylation