Improved microRNA cloning and analysis of RNA splicing

Abstract

MicroRNAs are single stranded RNA molecules of 19-25 nucleotides in length that are evolutionary conserved. MicroRNAs anneal through complementarity to messenger RNAs and can cause either inhibition of target messenger RNA translation or promotion of messenger RNA degradation. Mutations of miRNA genes can disrupt normal development and many other physiological processes.

IsomiRs are miRNA variants that are generated by imprecise Drosha or Dicer cleavage, many of these variants would be expected to target new messenger RNA and so may be of biological importance. We previously demonstrated that the expression of miRNAs and isomiRs can differ significantly between tissues as shown by cloning and sequence analysis. Here we used a bioinformatics approach to screen cell lines for markedly different ratios of miRNA:isomiR production, in order to establish model systems to investigate isomiR function.

We confirmed that the first step in the cloning of miRNAs by the commonly used Illumina protocol is biased but could be remedied by changing the condition of the ligation reaction and by the use of variable bases at the end of the 3’ adaptor RA3. We found that similar modifications could not reduce bias in the second step of cloning miRNAs to the standard 5’ RNA adapter RA5. We confirmed that the second step of cloning could be improved by using a combined RA3:RA5 adapter suggested to us by Somagenics. This required the successful identification of a suitable ligation enzyme and a reversible block for the 3’ end of adapter RA3:RA5. Our preliminary results indicate how the Somagenics protocol could be further improved.

In a second bioinformatics project we show how a sequencing database of spliced mRNA called Snaptron can be used to predict the effect of splice site mutations upon cryptic splice site activation and exon skipping, using BRCA1 and BRCA2 splice site mutations for the initial analysis. We discuss that this empirical approach compares favourably to in silico methods and is also of value for the analysis of other types of splicing mutations.