RNA Biology A brave new world with an ancient origin

1. RNA Biology A brave new world with an ancient origin

2. Genome Cellular function: Growth Differentiation maintenance, etc

3. Genome RNA Cellular function: Growth Differentiation maintenance, etc

4. Genome RNA Proteins Cellular function: Growth Differentiation maintenance, etc

5. The information content of the human genome (~25,000) 7% not transcribed } Protein-coding Capacity of the Human genome 1% ORF 1%UTR All snoRNAs 30-50% of miRNAs (~70,000) 35-40% Intron Non-protein-coding RNAs The Human Genome ENCODE Consortium (Nature 2007 Vol 447: 799-816)

6. Characterization of the anti-apoptotic activity of a TGFb responsive non-coding RNA The information content of the human genome 7% not transcribed 1% ORF (~25,000) 1%UTR All snoRNAs 30-50% of miRNAs (~70,000) 35-40% Intron Non-protein-coding RNAs The Human Genome ENCODE Consortium (Nature 2007 Vol 447: 799-816)

7. Mattick et al., 2006

8. Genome RNA Coding RNAs (mRNA) Non-coding RNAs Proteins Cellular function: Growth Differentiation maintenance, etc

9. Genome RNA Coding RNAs (mRNA) Ribosomal RNA snRNAs snoRNAs 7SK tRNA Regulatory RNAs: small and large Telomerase RNA Vault RNA SRP RNA ……… Non-coding RNAs Proteins Cellular function: Growth Differentiation maintenance, etc

10. Genome RNA Most abundant Ribosomal RNA snRNAs 7SK tRNA snoRNAs Regulatory RNAs: small and large Telomerase RNA Vault RNA SRP RNA ……… Coding RNAs (mRNA) Non-coding RNAs Proteins Cellular function: Growth Differentiation maintenance, etc

11. Genome RNA Most diverse Ribosomal RNA snRNAs snoRNAs 7SK tRNA Regulatory RNAs: small and large Telomerase RNA Vault RNA SRP RNA ……… Coding RNAs (mRNA) Non-coding RNAs Proteins Cellular function: Growth Differentiation maintenance, etc

12. Genome Introns RNA UTRs as Regulatory elements Ribosomal RNA snRNAs 7SK tRNA snoRNAs Regulatory RNAs: small and large Telomerase RNA Vault RNA SRP RNA ……… Coding RNAs (mRNA) Non-coding RNAs Proteins Cellular function: Growth Differentiation maintenance, etc

13. Earth, 4,000,000,000 years ago RNA world: RNA as catalyst and information carrier RNA enzymes (ribozymes)

14. Duties of a “living” ribozyme Information storage Replication Housekeeping functions

15. RNA side chains Adenine Guanine Cytidine Uracil Protein side chains

16. Protein Protein Proteins as slaves: the RNP world Information storage Replication Housekeeping functions

17. Protein Protein Proteins as slaves: the RNP world Information storage Replication Housekeeping functions

18. OH OH H H H CH3 CH3 DNA RNA

19. Protein Protein RNA Protein Information storage DNA Messenger RNA DNA DNA Replication Housekeeping functions

20. Protein RNA Non-coding Protein Information storage DNA Messenger RNA DNA DNA Replication Housekeeping functions Protein Regulation

21. C A 4 3 5 N G U 2 Phosphate Ribose (sugar) 6 N 5’ 1 Pyrimidine nucleobases Purine nucleobases 6 4’ 1’ 7 5 N 1 N 2’ 3’ 8 All numbers have primes 2 N 4 N 9 3 RNA is made up of sugar, nucleobase and phosphate

22. C A G U Phosphate Ribose (sugar) Pyrimidine nucleobases Purine nucleobases To make a nucleotide: linking the three together X X X Ester bond X X X X N-glycosidic bond

23. X X X C A X X X X G U Phosphate Ribose (sugar) Pyrimidine nucleobases Purine nucleobases To make a nucleotide: linking the three together Adenosine Monophosphate (AMP)

24. 6 7 5 N 1 N 8 2 N 4 N 9 3 4 3 5 N 2 6 N 5’ 1 4’ 1’ 2’ 3’ All numbers have primes Nomenclature of nucleic acids X X 3’-5’ phosphodiester

25. 6 7 5 N 1 N 8 2 N 4 N 9 3 4 3 5 N 2 6 N 5’ 1 4’ 1’ 2’ 3’ All numbers have primes Nomenclature of nucleic acids G Exocyclic amine A

26. 2’OH can easily cleave the RNA backbone base :

27. Watson-Crick or canonical basepairing G C U A

28. Watson-Crick or canonical basepairing G C Watson-Crick edges U A

29. Watson-Crick or canonical basepairing G C Watson-Crick edges U A Sugar edge/minor groove edge

30. Watson-Crick or canonical basepairing Major groove/Hoogstein edge G C Watson-Crick edges U A Sugar edge/minor groove edge

32. DNA B form helix RNA A form helix

33. Electronegativity of 2’ position substitution determines the sugar pucker

34. RNA STRUCTURE, FOLDING and DYNAMICS- AN OVERVIEW RNA can fold into compact structures capable of molecular recognition and catalysis RNA folding (and thus, function) depends on the association of numerous cations which act to 1. screen negative charge by electrostatic binding and 2. organize specific tertiary structure by site-specific binding RNAs often mis-fold or form more than one stable fold (RNA can fold to form kinetically trapped structures as well as equilibrium structures with similar thermodynamic stabilities). RNA-protein interactions can involve large and small-scale conformational changes which permit the formation of very stable complexes with unique structures. RNA binding domains are highly diverse and RNA binding proteins can contain several that function in concert Protein binding can influence RNA folding resulting in regulation of function, ordered binding in complex ribonucleoproteins, enhanced molecular recognition (specificity), ????????? (some) BIOLOGICAL IMPLICATIONS: RNA has the highest functional potential of any macromolecule (the primordial stuff?) RNA structure (and thus function) can be highly sensitive to ligand binding- allows regulation Structural flexibility with protein allows formation of structures unattainable by RNA alone (kinetic and thermodynamic effects) (some) EXPERIMENTAL IMPLICATONS: Testing for the involvement of formation of structure with specific aspects of function is difficult Both kinetic and thermodynamic effects on folding must be considered High resolution structures may not necessarily reflect the functional conformation(s)

35. RNA can fold into compact structures capable of molecular recognition and catalysis -RNA structure is hierarchical -Helical domains associate into bundles, tertiary structure is maintained by long-range non-covalent interactions. -Bases in loops, bulges and junctions are most often paired, but usually form non-canonical base pairs -A-form RNA helices are not well suited to undergo specific interactions because the major groove is narrow and deep and the minor groove does not display sequence specificity. -Non-canonical base pairs widen the major groove, thereby making it accessible to ligands -Large RNAs are often composed of several structural domains, which can assemble and fold independently -Phylogenetically conserved features form the core of functional structure, variable regions are typically located on the periphery where they stabilize folding

36. A) single stranded regions B) duplex C C) hairpin D) internal loop D E) bulge loop G E F F) junction B A G) pseudoknot RNA structure Primary structure formed by unpaired nucleotides Secondary structure double helical RNA (A-form with 11 bp per turn) duplex bridged by a loop of unpaired nucleotides nucleotides not forming Watson-Crick base pairs unpaired nucleotides in one strand,other strand has contiguous base pairing DNA RNA Mutations Amino acids, protein structure three or more duplexes separated by singlestranded regions tertiary interaction between bases of hairpin loopand outside bases

37. RNA structure How to predict RNA secondary/tertiary structure? Probing RNA structure experimentally: - physical methods (single crystal X-ray diffraction, electron microscopy) - chemical and enzymatic methods - mutational analysis (introduction of specific mutations to test change in some function or protein-RNA interaction) Thermodynamic prediction of RNA structure: - RNA molecules comply to the laws of thermodynamics, therefore it should be possible to deduce RNA structure from its sequence by finding the conformation with the lowest free energy - Pros: only one sequence required; no difficult experiments; does not rely on alignments - Cons: thermodynamic data experimentally determined, but not always accurate; possible interactions of RNA with solvent, ions, and proteins Comparative determination of RNA structure: - basic assumption: secondary structure of a functional RNA will be conserved in the evolution of the molecule (at least more conserved than the primary structure); when a set of homologous sequences has a certain structure in common, this structure can be deduced by comparing the structures possible from their sequences - Pros: very powerful in finding secondary structure, relatively easy to use, only sequences required, not affected by interactions of the RNA and other molecules - Cons: large number of sequences to study preferred, structure constrains in fully conserved regions cannot be inferred, extremely variable regions cause problems with alignment DNA RNA Mutations Amino acids, protein structure

38. RNA folding (and thus, function) depends on the association of numerous cations Cation binding acts to screen negative charge by electrostatic binding and to organize specific tertiary structure by site-specific binding “Diffuse ions” accumulate near the RNA because of the RNA electrostatic field and remain largely hydrated. A “chelated” or “site-bound” ion directly contacts a specific location on the RNA surface and is held in place by electrostatic, H-bonding and coordination interactions. diffuse ions are a major factor in the stabilization of RNA tertiary structures.

39. As a negatively charged polyelectrolyte, RNA attracts a diffuse counter ion atmosphere. In addition, site-bound Mg(2+) (and also monovalent cations) can serve as specific cofactors to stabilize functional RNA folds and divalent ions can act directly in RNA catalysis.

40. The structures of RNA-binding proteins are diverse Protein domains have evolved to bind ssRNA, dsRNA and many recognize complex tertiary structure

41. Surface electrostatic calculations and shape determine the interactions RNAs can participate in

42. RNA Structure and Function: BIOC 599 Course structure: 3 sessions per topic: Lecture by instructor Literature discussion by instructor Literature discussion by student Evaluation mechanism: Participation in class discussion: 33% Quality of presentation: 33% Mini-grant: 33% 1% guaranteed 2 absences/semester Don’t be late! Plagiarism: don’t cut and paste, rephrase!

43. Criteria for evaluation of presentations The instructors will be grading you based on the below criteria: The student had sufficient background knowledge The student had read the supplementary data (if any) The student could place the paper in the big picture The student clearly understood why the experiments were done The student understood the techniques used in the paper The student could summarize the paper and discuss its significance The student was successful in critically evaluating the paper The student suggested extra experiments/future directions The slides were clear and well-organized The presentation included sufficient background material The presentation was coherent and clear

44. Criteria for evaluation of minigrants The grant was clearly-written and was well-organized The grant placed the scientific question posed in the big picture The grant was hypothesis driven It contained sufficient background material The experimental design was clear and well-reasoned The experiments followed a logical line of reasoning The grant addressed a significant, major question left unaddressed by the paper presented in the class The experiments were indeed the logical follow up to the paper presented in class The experiments were innovative and sufficiently complex, rather than a simple repeat of the experiments in the paper with minor changes or very simple, obvious experiments The student had considered the possibility that the proposed experiment might not work, and had listed alternative strategies for each major step There were validation steps included for major experiments, if applicable The proposed experiments seemed feasible The grant contained clear interpretation of the expected experimental results Common pitfalls were listed for each experiment Sufficient experimental detail was included, at the level commonly put in materials and methods section of papers The grant contained a statement indicating what the contribution of the proposed experiments will be to the field in general.