Sequence determination of rat U5 RNA using a chemical modification procedure for counteracting sequence compression

Area of Science:

• Molecular Biology
• RNA Biochemistry

Background:

• U5 RNA is a component of the spliceosome, crucial for pre-mRNA splicing.
• Understanding U5 RNA's structure and sequence is vital for elucidating spliceosome function.

Purpose of the Study:

• To determine the complete nucleotide sequence of a major rat liver U5 RNA species.
• To characterize the modified nucleotides and microheterogeneity within U5 RNA populations.
• To develop a method to resolve sequence compression artifacts in gel electrophoresis.

Main Methods:

• Post-labeling techniques for nucleotide sequencing.
• Chemical modification using sodium bisulfite and formamide hydrolysis.
• Polyacrylamide gel electrophoresis in 7 M urea.

Main Results:

• The nucleotide sequence of the major rat liver U5 RNA species (117 nucleotides) was elucidated: XpppAmUmACUCUGGUUUCUCUUCAGAUCGUAUAAAUCUUUCGmCCUUmUpsiACmNAAAGAUpsiUCCGUGGAGAGGA ACAACUCUGAGUCUUAAACCAAUUUUUUGAGGCCUUGUCUUGA(G)CAAGGCUOH.
• Identified modified nucleotides including a 5'-cap structure, Gm, Um, psi, Cm, and N.
• Observed microheterogeneity with U5 RNA species lengths of 117, 118, and 119 nucleotides.
• Developed a chemical modification procedure to denature secondary structures and overcome sequence compression during electrophoresis.

Conclusions:

• The complete sequence and modified nucleotide content of rat liver U5 RNA were established.
• A novel chemical treatment effectively resolves sequence compression in gel electrophoresis, improving RNA sequencing accuracy.
• This study provides a robust method for analyzing RNA sequences affected by secondary structure.