Abstract
Eukaryotic messenger RNA (mRNA) is modified by the addition of an inverted guanosine cap to the 5′ triphosphate. The cap guanosine and initial transcribed nucleotides are further methylated by a series of cap methyltransferases to generate the mature cap structures which protect RNA from degradation and recruit proteins involved in RNA processing and translation. Research demonstrating that the cap methyltransferases are regulated has generated interest in determining the methylation status of the mRNA cap structures present in cells. Here, we present CAP-MAP: cap analysis protocol with minimal analyte processing, a rapid and sensitive method for detecting cap structures present in mRNA isolated from tissues or cultured cells.
Highlights
Eukaryotic RNA polymerase II (RNAPII)-transcribed RNAs are modified by the addition of 7-methylguanosine to the 50 triphosphate found on the first transcribed nucleotide, forming the cap structure denoted m7GpppN (N is any nucleotide) [1,2]
We developed CAP-MAP to detect and quantify the messenger RNA (mRNA) cap structures present in cells and tissues
The method assesses the permutations of N-7 methylation of the terminal cap guanosine, O-2 methylation of the first nucleotide ribose and N-6 methylation of the first nucleotide adenosine in mRNA caps from biological samples
Summary
Eukaryotic RNA polymerase II (RNAPII)-transcribed RNAs are modified by the addition of 7-methylguanosine to the 50 triphosphate found on the first transcribed nucleotide, forming the cap structure denoted m7GpppN (N is any nucleotide) (figure 1) [1,2]. For many short RNAPII transcripts involved in guiding RNA processing and modification, the cap is a precursor for further modification [3]. For pre-messenger RNA (mRNA), the cap guides transcript processing and selection for translation via interactions with cap-binding proteins, while protecting the transcript from 50–30 exonucleases [4]. Metazoan mRNA caps contain ribose methylated on the O-2 position of the first and second transcribed nucleotides (denoted Nm), which creates a further ‘self-mRNA’ mark, enabling innate immunity proteins to differentiate it from unmethylated, foreign RNA [5,6]. Decapping is regulated by RNA binding proteins which promote or antagonize the recruitment of decapping complexes to the mRNA [7]. The cap is essential for the proper processing, function and lifespan of an mRNA
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
