1. Introduction to Bioinformatics - Tutorial no. 9 RNA Secondary Structure Prediction

2. Rfam http://www.sanger.ac.uk/Software/Rfam Rfam is a large collection of multiple sequence alignments and covariance models covering many common non-coding RNA families. For each family in Rfam you can:  View and download multiple sequence alignments  Read family annotation  Examine species distribution of family members  Follow links to other databases

3. Rfam Browse Browse the Rfam hierarchy

4. Rfam Search Search by EMBL ID Search your own sequence

5. RNA structure prediction Vienna RNA package:  RNAfold -- predict minimum energy secondary structures and pair probabilities RNAfold  RNAalifold – predict consensus secondary structure  RNAeval -- evaluate energy of RNA secondary structures RNAeval  RNAheat -- calculate the specific heat (melting curve) of an RNA sequence RNAheat  RNAinverse -- inverse fold (design) sequences with predefined structure RNAinverse  RNAdistance -- compare secondary structures RNAdistance  RNApdist -- compare base pair probabilities RNApdist  RNAsubopt -- complete suboptimal folding RNAsubopt Web interface: to RNAfold, RNAalifold, RNAinverse Other can be downloaded for Unix and for Windows.

6. RNAfold : http://www.tbi.univie.ac.at/~ivo/RNA/ Gives best stabilized structure (structure with mfe – minimal free energy) In addition, uses a partition function and base pair probabilities in the thermodynamic ensemble (default and recommended).

7. Input (sequence only) Fold Algorithm RNA or DNA parameters Target temperature Advanced fold options Output formats Link to your previous run Email (necessary for large sequences)

8. Output in bracket notation Output - PostScript

10. Free energy (∆G) Enthalpy (∆H) Melting (de-hybridization) temperature

11. RNAalifold: Predicts consensus secondary structures for sets of aligned RNA (ClustalW files). Information from the alignment: 1.Conserved nucleotide pairs are shown normally. 2.Pairs with consistent mutations, which support the structure, are marked by circles. 3.Pairs with inconsistent mutations are shown in two shades of gray.

12. . - unpaired base ( ) - base i pairs base j {} - a weaker version of the above | - a base that is mostly paired but has pairing partners both upstream and downstream Bracket notation: (((..((((...)))).))) =

13. Question Indicate which of the structure represent the same secondary structure

14. Question Yossi is a brilliant student in the “Introduction to Bioinformatics” course with a great gut feeling. The moment, Yossi saw the following genomic sequence, he understood that it contains a functional RNA. Yossi checked his proposal and discovered that indeed he was right, however when looking at the structure he suddenly realized that something is wrong…… To help Yossi:  Repeat the experiment that Yossi ran to check his predictions.  Assuming a single mutation had accord in the sequence, find the mutation that caused the unexpected results.  Correct this problem and present the correct results.

15. tRNAscan

16. Question (cont.) The problem is the internal loop in the stem of the right leaf. Using tRNAscan tool, we can find that the given sequence contains the tRNA between bases 248 and 324. Running RNAfold, we receive the following structure:

17. Question (cont.) Running Blastn we can locate the mutation A->C at position 299 of the given sequence. The corrected structure: