1. http://creativecommons.org/licenses/by-sa/2.0/ Mirela Andronescu
February 22, 2005
Lab 8.3
(c) 2005 CGDN

2. RNA LAB Mirela Andronescu (UBC)

3. Lab 8.3: RNA – Outline RNA Databases Secondary structure prediction
Mirela Andronescu
February 22, 2005
Lab 8.3: RNA – Outline
RNA Databases
Secondary structure prediction
Structure visualization (tertiary)
Prediction of consensus secondary structure
Searching homologues in genomes
Lab 8.3
(c) 2005 CGDN

4. Setup You’ll do 7 activities, each with tasks and questions
Some of the activities are in a browser, some at the command prompt
Open a terminal
Check if you have the directory rnalab
If you don’t, download from the course web page
Lab 8.3

5. Activity 1 Downloading RNA secondary structures from Gutell database
Mirela Andronescu
February 22, 2005
Activity 1
Downloading RNA secondary structures from Gutell database
What is Gutell database?
Gutell online database contains homologous RNA sequences (mostly ribosomal RNA)
Some have associated secondary structures, very accurately determined through comparative sequence analysis
Lab 8.3
(c) 2005 CGDN

6. A1: Gutell DB Open a browser and go to Gutell DB site
Follow Gutell link on the course web page
Or type
Login with cbw2005/rnalab
Lab 8.3

7. Lab 8.3

8. A1: Gutell DB On top, click on tab 3. Sequence and structure data
Then on left, click on A. INDEX of Available RNA Sequences and Structures
Click on the 24 representing 5S rRNA structures of Bacteria
Lab 8.3

9. Lab 8.3

10. A1: Gutell DB View a PS or PDF file Select PS or PDF on top
Click on the first link in the column StrDiags
Lab 8.3

11. Lab 8.3

12. Lab 8.3

13. A1: Gutell DB Save a bpseq file Select bpseq on top
Click on the first link in the column StrDiags
Save the file into the directory rnalab
Lab 8.3

14. Lab 8.3

15. Lab 8.3

16. Activity 1 question
What is the length of the structure you’ve just viewed and downloaded? (without counting)
Tip: the last line of the bpseq file will give you the answer
Lab 8.3

17. Activity 2
Predicting RNA secondary structures using RNAfold from Vienna RNA package
What is RNAfold?
RNAfold is a program which predicts RNA secondary structure from sequence, using a dynamic programming algorithm
A very similar and popular program is mfold
Accuracy is about 73% on average
Lab 8.3

18. A2: RNAfold
Extract the sequence out of the bpseq file you have downloaded from Gutell DB
You’ll use a simple Perl script: bpseq2seq.pl
You could write such a script yourself
This script is in the rnalab directory, you can read it
At the command prompt, type:
./bpseq2seq.pl d.5.b.A.tumefaciens.bpseq > gutell.txt
gutell.txt contains an RNA sequence
Lab 8.3

19. A2: RNAfold Run RNAfold software At the command prompt, type:
RNAfold < gutell.txt
The predicted structure in dot-parenthesis format is displayed, as well as the predicted minimum free energy
Lab 8.3

20. Activity 2 questions What is the predicted minimum free energy?
Visualize the predicted structure
At the command prompt, type:
gv rna.ps &
How many multi-loops there are in this structure?
Tip: the following structure has one multi-loop
Lab 8.3

21. Multi-loop with
three branches
Lab 8.3

22. Activity 3
Downloading a tertiary RNA structure from PDB (Protein Data Bank)
What is PDB?
PDB is a database containing tertiary structures of proteins and RNAs, determined by NMR or X-ray
Lab 8.3

23. A3: PDB Open a browser and go to PDB database
Follow the link on the course web page
Or type as address
Type 1C2X in the search box
Lab 8.3

24. Lab 8.3

25. A3: PDB Click Download/Display File
Click the TEXT link for complete PDF file
Click Save full entry to disk and save
Lab 8.3

26. Lab 8.3

27. Lab 8.3

28. Lab 8.3

29. Activity 3 questions Search pseudoknotted RNA structures in PDB
Tip: type RNA pseudoknot in the search box on the first page of PDB
Save to disk a PDB file in the list
What is the PDB ID of the structure you chose?
Lab 8.3

30. Activity 4 Visualizing tertiary RNA structures using RasMol
What is RasMol?
RasMol is a visualization tool for tertiary structures (proteins or RNA)
You can see each atom
You can rotate the figure with the mouse
Takes as input a PDB file
Lab 8.3

31. A4: RasMol display
Lab 8.3

32. A4: RasMol At the command prompt, type: View the structure
rasmol 1C2X.pdb &
View the structure
Rotate the structure with the mouse
Lab 8.3

33. Activity 4 questions
Can you see the structure you are viewing is similar to the secondary structure in the next slide?
Visualize with RasMol the pseudoknotted structure you downloaded at the end of Activity 3.
Lab 8.3

34. Lab 8.3

35. Activity 5 Predicting a consensus structure using Alidot
What is Alidot?
Alidot takes as input a set of homologous sequences and their minimum free energy secondary structure predicted with RNAfold
Predicts the consensus structure
Lab 8.3

36. A5: Alidot
This activity is to be performed at the command prompt, using files in your rnalab directory
The file bact5s.seq contains 13 input homologous sequences representing 5S rRNA, all from Gutell database
You can read the information in this file
Lab 8.3

37. A5: Alidot First align these sequences. At the command prompt type:
clustalw bact5s.seq
This will create the file bact5s.aln
Fold all these sequences with RNAfold
RNAfold -p < bact5s.seq > bact5s.fold
(-p uses the partition function)
Lab 8.3

38. A5: Alidot
The output file bact5s.fold contains all folded sequences, but Alidot wants them in separate files:
split.pl bact5s.fold (don’t use split!!)
Finally, run Alidot, which uses the structure files just created, and the alignment:
alidot < bact5s.aln > alidot.out
Create a figure with the consensus structure:
cons.sh
Lab 8.3

39. Activity 5 questions Visualize the consensus structure
gv bact5s.ps &
How many conserved stems there are?
Try to find the corresponding stems in the structure from Gutell DB that we viewed in activity 1 (see next slide).
Lab 8.3

40. Lab 8.3

41. Activity 6
Searching homologous sequences in a genome fragment, using Infernal
What is Infernal?
Infernal searches sequences in a genome, which are homologous to a covariance model
Why? To look for conserved/functional elements
Infernal also creates structure-based multiple sequence alignments
Lab 8.3

42. A6: Infernal diagram alignment w/ sec. str. annotation covariance
model
cmbuild
covariance model +
genome or DNA seq
cmsearch
hits
genomes or DNA seqs
cmalign
structural
alignment
Lab 8.3

43. A6: Infernal
This activity is to be performed at the command prompt, using files in your rnalab directory
The file RNAI.sto contains 10 RNAI (RNA Interference) homologous sequences from different organisms. They are aligned and they have a consensus structure associated
Lab 8.3

44. A6: Infernal
Given the file RNAI.sto, build a covariance model RNAI.cm:
cmbuild RNAI.cm RNAI.sto
output
input
Lab 8.3

45. A6: Infernal
The file RNAI.db contains a genome fragment from another organism, which contains an RNAI gene
Search for RNAI homologues in this fragment
cmsearch RNAI.cm RNAI.db
input
Lab 8.3

46. A6: Infernal
The file RNAI.fa contains several genome fragments from various organisms
Create a structure-based multiple sequence alignment of these fragments
cmalign RNAI.cm RNAI.fa
input
Lab 8.3

47. Activity 6 questions How many hits did you find with cmsearch?
Watch the structure-based alignment returned by cmalign
Compare with the alignment obtained with clustalw (see next 2 slides)
Do the two alignments differ a lot?
Lab 8.3

48. Lab 8.3

49. Activity 7 Browsing RNAI in Rfam database What is Rfam?
Rfam is a database of RNA families of homologous non-coding RNA
Rfam was built using Infernal. Starting from a set of aligned sequences with known structure (called “seed”), new homologous sequences were found and aligned.
Lab 8.3

50. A7: Rfam Open a browser and go to Rfam database
Follow the Rfam link on the course web site
Or type
Click on the tab Browse Rfam
Expand Gene, then antisense
Click RNAI – RNAI
Lab 8.3

51. Lab 8.3

52. Lab 8.3

53. A7: Rfam
This will open a page with information about RNAI, a consensus structure, and a table
The column Alignment allows download of aligned sequences
The file RNAI.sto that you used as input to Infernal was obtained from here
The column Member sequences opens a list of sequences, with the EMBL accession numbers
Lab 8.3

54. Lab 8.3

55. Lab 8.3

56. Activity 7 questions
Find the EMBL accession number of an RNAI which was used in the seed
How many sequences from the family U1 spliceosomal RNA does Rfam contain in total?
Tip: see the next two slides:
Click Browse Rfam
Expand Gene, then snRNA, then splicing
Click on U1 spliceosomal RNA
Read the value near Full under Member sequences
Lab 8.3

57. Lab 8.3

58. Lab 8.3

59. Recap Databases: Gutell, PDB, Rfam Visualization - tertiary: RasMol
Secondary structure prediction: RNAfold
Consensus structure prediction: Alidot
Searching homologous structures: Infernal
Lab 8.3