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Characterization of lariat RNAs in plants.

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posted on 21.11.2016, 18:00 by Ziwei Li, Shengpeng Wang, Jinping Cheng, Chuanbin Su, Songxiao Zhong, Qi Liu, Yuda Fang, Yao Yu, Hong Lv, Yun Zheng, Binglian Zheng

(A) Three examples of identified lariat RNAs. Normalized peaks of lariat RNAs are highlighted by black rectangles. The exon is boxed in blue, and the intron is a line. The X-axis indicates the chromosomal location. The Y-axis indicates normalized peaks from the genomic region. Reads counts were normalized to tag per 10 million (TP10M) to adjust for differences in sequencing depth of the two RNA-seq libraries. (B) Schematic of divergent primers for lariat RNAs and convergent primers for linear mRNAs. The purple and blue boxes indicate exons; A represents the branch point. (C) Validation of lariat RNAs by RT-PCR with the divergent PCR primer pairs shown in (B) in Col-0, dbr1-2, and Compl (pDBR1::DBR1-RFP dbr1-2) with or without RNase R treatment. UBQ5 was used as the loading control. (D) Validation of lariat RNAs by qRT-PCR using total RNA of Col-0, dbr1-2, and Compl (pDBR1::DBR1-RFP dbr1-2). The amount of lariat RNAs was normalized to UBQ5. Error bars show SE calculated from three biological replicates. (E) qRT-PCR showing resistance of lariat RNAs to RNase R digestion. Linear mRNAs (blue font indicated) are positive controls for RNase R treatments. The amount of RNAs after RNase R treatment was normalized to the RNase R-untreated sample. Error bars show SE calculated from three biological replicates.