Lower-stem-secondary-structure

Scripts used for the analysis of the pri-miRNA lower stem structure.

Genomic sequences of pri-miRNAs were obtained from UCSC Genome Browser. Each pri-miRNA analyzed consists of the pre-miRNA and flanking sequences of 30 nt both sides. The secondary structure was obtained using RNAfold (Gruber et al., 2008), and the bracket-dot notation of the lower stem was analyzed using this R scripts.

In summary, we first tested whether a nucleotide in one position is paired with another on the other side of the pre-miRNA. Nucleotides that failed such a test were labeled as unpaired. Then, we aligned all pri-miRNA 5p sequences by the 5’ Drosha cleavage site (5’ end of pre-miRNA) and aligned all 3p sequences by the 3’ Drosha cleavage site (3’ end of pre-miRNA). The fraction of paired nucleotides was calculated for each position and was plotted against its relative distance to the Drosha cleavage site.

Example 1

1. pri-miRNA secondary structure

Secondary structure from pri-miRNA sequence reported in miRBase 21 usually don't contain many upstream or downstream nucleotides from the Drosha cleavage sites.

>hsa-mir-9-1 MI0000466
CGGGGUUGGUUGUUAUCUUUGGUUAUCUAGCUGUAUGAGUGGUGUGGAGUCUUCAUAAAGCUAGAUAACCGAAAGUAAAAAUAACCCCA
.(((((((....((((.(((((((((((((((.((((((.(.(....).).)))))).))))))))))))))).))))...))))))).

Therefore, we obtained the secondary structure from pri-miRNA sequence obtained using RNAfold on longer genomic sequences on UCSC Genome Browser.

......(((((...(.(((((((....((((.(((((((((((((((.((((((.(.(....).).)))))).))))))))))))))).))))...))))))).)...))).)).......

Note that in most cases the secondary structure of the pri-miRNA sequence reported in miRBase21 is already included in the longer sequence. Most discrepancies might come from alternative foldings derived from providing extra-context to RNAfold.

2. Isolate the 5' and 3' lower stem segments

Structure of the 5' lower stem

-30__________________________-1
......(((((...(.(((((((....(((

Structure of the 3' lower stem

+1_________________________+30
))...))))))).)...))).)).......

3. Fraction of stem base-paired nucleotides

For each position on the lower stem, we count the frequency of nucleotides on the 5' with ( over the total, while on the 3' we count only the ).

Example 2

4. Detection of secondary stems

It is not unfrequent to detect pri-miRNA where the lower stem, due to its less structured nature, presents secondary stems.

hsa-mir-152 5' lower stem
-30__________________________-1
..((((...)))..))((((..((.(((((

In such cases, our script excludes those positions from the being counted as "lower stem base-paired" nucleotides. To that aim our code replaces in the bracket-dot notation, the brackets on the secondary stem for *.

hsa-mir-152 5' lower stem
-30__________________________-1
..***....***..**((((..((.(((((

Acknowledgements:

This code was written by Susanna Chen and supervised by Xavier Bofill-De Ros.

References:

Gruber, A.R., Lorenz, R., Bernhart, S.H., Neuböck, R., and Hofacker, I.L. (2008). The Vienna RNA websuite. Nucleic Acids Res 36, W70-4.