RNA secondary structure

Transcription

1 Sequence Analysis '17 -- Lecture 17 RNA secondary structure Functions Representations Predictions Many slides courtesy of M. Zuker, RP Math

2 When RNA secondary structure matters mrna --> protein protein ssrna Strong secondary structure can block translation.

3 especially sensitive is the... Anderson RBS family -- bacterial. RBS ribosome binding site fmet NNN NNNNAAAGA GA UUUCU CU NNNNNAUGNNNN NNNN 5' 3' 16s species specific! Registry of Standard Biological Parts

4 microrna (mir) Found in 3'UTR introns exons transcription, folding nucleus cytoplasm microrna primary-microrna passenger strand degraded dicer argonaut proteins microrna duplex blocks translation deadenylation endonuclease digestion

5 Ambiguous bases

6 RNA secondary structure is base pairing i j Rules for normal SS

7 Not just Watson-Crick.. Different ways of base-pairing allow RNA to adopt duplex structures beyond A and B helix. f any base-pair is possible, how do we predict pairings?

8 2D plot M B M Structure types within RNA sec struct E M B

9 B M M E M B note: No bp lines cross.

10 Converting from 2D plot sec struct to circle/tree.

11 Pseudoknots C G A G G GU Circle plot=====> C UC U C C C C A G G C A U C

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16 Prediction of RNA secondary structure Comparative methods, phylogenetics Free energy calculations Dot plot - Easy. - Can be done on a single sequence. - Cannot find non-canonical base pairs. Mutual information (M) - Requires a deep multiple sequence alignment - Can find non-canonical base-pairs.

17 Comparative modeling assume conserved structure between homologs

18 RNA structure by energy minimization Assumes energy is the sum of base pairs. where, e(i,j) = 0 if j-i < 4 Forward summation of energy matrix E: too close, can't pair add to loop add to loop start a helix, or add a base pair join helices in multi-loop

19 RNA structure by energy minimization 2. base-pairing is found by tracing back from (1,n) Stack A becomes the answer: a list of base pairs Stack B is a list of unfinished segments energy matrix 1. energy matrix is initilized starting from diagonal.. Find k such that E i,j = E i,k + E k+1,j Push (i,k) and (k+1,j) onto Stack B. Stop with error if no such k exists.

20 RNA structure by Dot Plot

21 RNA structure by Dot Plot

22 RNA structure by Mutual nformation ow-to: 1) Run BLAST search to get homologs. 2) Prune sequences to remove redundancy.* 3) Prune columns to remove uninformative data. (conserved positions tell you nothing) 4) Calculate mutual information (M i,j ) for all pairs of positions (i,j). position position position

23 Mutual information, in general A measure of the surprisingness of a pair of events. frequency of a pair of events observed together= N(a,b events)/n(total events) in bits, because we used log2 M = Σ f(a,b) log 2 ( ) f(a,b) f(a)f(b) a,b {events} sum over all event pair types expected frequency of a,b events together is the product of the frquencies of the events separately

24 Mutual information for base-pairs A measure of the surprisingness of the evolution of two positions in the sequence. species i position j pair of sequence positions (i,j) frequency of base-pair (B 1, B2) at positions (i,j) =N(B1,B2)/N(total sequences) M(i,j) = Σ ( f i,j (B 1,B 2 ) ) f i,j (B 1,B 2 ) log 2 f i (B 1 )f j (B 2 ) B 1,B 2 {A,C,G,T} sum over all base-pair types expected frequency of base B1, B2 Exercise: Calculate M(i,j)=

25 Can you find pairs of positions with high M? W-C non-canonical

26 Mutual information matrix for 20 aligned sequences very noisy. Take home message: You need lots of sequence to do mutual information analysis. And this is for RNA where the signal is strong. Try protein. You'll need thousands of sequences...

27 Same RNA, M i,j for 302 aligned sequences cleaner

28 Corresponding structure. M elices now appear as straight lines of dots, after re-numbering to remove gaps in the MSA

29 Prediction of RNA secondary structure Comparative methods, phylogenetics Free energy calculations Dot plot - Easy. - Can be done on a single sequence. - Cannot find non-canonical base pairs. Mutual information (M) - Requires a deep multiple sequence alignment - Can find non-canonical base-pairs.

30 Review questions? 1. What are the UPAC codes? 2. What are the different types of RNA structure? 3. What is mutual information? ow is it calculated? 4. What are the sources of energy for RNA structure? 5. What algorithm is used to calculate the energy over all RNA structures? 6. What is expressed in a dot plot? 7. What is expressed in a circle plot? 8. What is a pseudoknot? 9. What is a non-canonical basepair? 10. Can you convert a dotplot into a graph? 11. Can you convert a graph into a circle plot? 12. Can you see a pseudoknot in a graph, circle, dotplot?