Unraveling 3'-end RNA uridylation at nucleotide resolution

Abstract

Post-transcriptional modification of RNA, the so-called 'Epitranscriptome', can regulate RNA structure, stability, localization, and function. Numerous modifications have been identified in virtually all classes of RNAs, including messenger RNAs (mRNAs), transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), microRNAs (miRNAs), and other noncoding RNAs (ncRNAs). These modifications may occur internally (by base or sugar modifications) and include RNA methylation at different nucleotide positions, or by the addition of various nucleotides at the 3'-end of certain transcripts by a family of terminal nucleotidylyl transferases. Developing methods to specifically and accurately detect and map these modifications is essential for understanding the molecular function(s) of individual RNA modifications and also for identifying and characterizing the proteins that may read, write, or erase them. Here, we focus on the characterization of RNA species targeted by 3' terminal uridylyl transferases (TUTases) (TUT4/7, also known as Zcchc11/6) and a 3'-5' exoribonuclease, Dis3l2, in the recently identified Dis3l2-mediated decay (DMD) pathway - a dedicated quality control pathway for a subset of ncRNAs. We describe the detailed methods used to precisely identify 3'-end modifications at nucleotide level resolution with a particular focus on the U1 and U2 small nuclear RNA (snRNA) components of the Spliceosome. These tools can be applied to investigate any RNA of interest and should facilitate studies aimed at elucidating the functional relevance of 3'-end modifications.

Keywords: 3′-end Uridylation; DIS3L2; DIS3L2-mediated decay (DMD); TUTase; U1; U2; snRNA.

Figure 1.

cRACE and 3?-ligation protocols. Schematic representation of cRACE (A) and 3?-ligation RACE (B) methods are shown. (C) Ethidium bromide staining of PCR products for U1 and U2 snRNAs (3?-ligation RACE) run on 1% agarose gel. Dashed rectangles represent the regions that were cut and used for DNA extraction from the gel. White arrowheads point to elongated slow-migrating PCR product accumulated especially in IP samples.

Figure 2.

Flowchart of the TailSeq function.

Figure 3.

3?-ligation analysis of Dis3l2-bound U1 snRNA. (A) Pie chart representing the percentage of the sequencing reads containing only 1 primer and 1 adapter. Only 4 percent of the total reads contained more than 1 primer of adapter. (B) Bar diagram representing the percentage of the reads in input and Dis3l2-IP with indicated genomic extension (plus numbers), truncation (minus numbers), or annotated ends (zero). (C) Bar diagram showing the number of reads in input and Dis3l2-IP with indicated length of U-tails. (D) Logistical regression of U-tail existence depending on the length of the genomic extension and the respective probabilities (right panel).

Figure 4.

3?-ligation analysis of Dis3l2-bound U2 snRNA. (A) Pie chart representing the percentage of the sequencing reads containing only 1 primer and 1 adapter. Note the negligible portion of the reads with more than 1 primer or adapter. (B) Bar diagram representing the percentage of the reads in input and Dis3l2-IP with indicated genomic extension (plus numbers), truncation (minus numbers), or annotated ends (zero). Note the increased extended reads especially in the IP sample. (C) Distribution of the sequencing reads in input and Dis3l2-IP with indicated length of U-tails. (D) Logistical regression of U-tail existence depending on the length of the genomic extension and the respective probabilities (right panel).