Chapter 3: The Versatility of RNA

1. Chapter 3: The Versatility of RNA

2. The most surprising aspect of all of this is how late in the study of cell biology the importance and ubiquitous nature of RNA in gene regulation became widely recognized. Philip Sharp, Cell (2009), 136:580

3. 3.1 Introduction

4. RNA is a modular structure built from a combination of secondary and tertiary structural motifs. • RNA chains fold into unique 3-D structures, which act similarly to globular proteins. • RNA is involved in a wide range of cellular processes from DNA replication to protein synthesis.

5. 3.2 RNA is involved in a wide range of cellular processes

6. Six major types of RNA • Ribosomal RNA (rRNA) • Messenger RNA (mRNA) • Transfer RNA (tRNA) • Small nuclear RNA (snRNA) • Small nucleolar RNA (snoRNA) • MicroRNA (miRNA)

7. Two key points for understanding RNA function • RNA can form complementary base pairs with other nucleic acids. • RNA can interact with proteins: Ribonucleoprotein (RNP) particles

8. Overview of the versatility of RNA • RNA can serve as a “scaffold” upon which proteins can be assembled. e.g. signal recognition particle (Chapter 14) • RNA-protein interactions can influence the catalytic activity of proteins. e.g. telomerase (Chapter 6) • RNA can be catalytic (this Chapter). • Small RNAs can directly control gene expression (Chapter 13). • RNA can be the hereditary material (this Chapter).

9. 3.3 Structural motifs of RNA

10. Secondary structure of RNA • RNA is a chain-like molecule composed of subunits called nucleotides joined by phosphodiester bonds. • Each nucleotide subunit is composed of a ribose sugar, a phosphate group, and a nitrogenous base. • The common bases in RNA are adenine (A), guanine (G), cytosine (C), and uracil (U).

11. Secondary structure: the folding of an RNA chain into a variety of structural motifs. • Common secondary structures can be predicted by computer analysis.

12. Base-paired RNA adopts an A-type double helix • 2'-OH group hinders formation of a B-type helix. • Shallow, broad minor groove includes the ribose 2'-OH. • RNA is often recognized by RNA-binding proteins in the minor groove.

13. RNA helices often contain noncanonical base pairs • >20 different types of noncanonical (non-Watson-Crick) base pairs. • Widen the major groove and make it more accessible to ligands or proteins. • The most common of these are the: GU wobble GA sheared AU reverse Hoogsteen GA imino

14. RNA structures often contain unconventional base pairing Base triples • Typically involve one standard base pair. • The third base interacts in a variety of unconventional ways.

15. Noncanonical base pairs and base triples Important mediators of: • RNA self-assembly. • RNA-protein interactions. • RNA-ligand interactions.

16. “tRNA looks like Nature’s attempt to make RNA do the job of a protein.” Francis Crick (1966) Cold Spring Harbor Symposium on Quantitative Biology 31:1-9.

17. tRNA structure: important insights into RNA structural motifs General principles • Modified bases. • tRNA loops each have a separate function. • Coaxial stacking of stems.

18. Pre-tRNA transcript is processed at both the 5′ and 3′ ends. • The average tRNA is about 76 nt long. • All tRNAs fold into the same general shape. Cloverleaf secondary structure L-shaped tertiary structure

19. Modified bases • More than 50 modified bases in tRNA. • Simple methylation to complete restructuring of the purine ring. • Inosine (I) was the first modified nucleoside to be identified in tRNA. • Pseudouridine () was the first identified in any RNA. • Extensive base modification also occurs in other RNAs, e.g. during maturation of ribosomal RNA.

20. tRNA loops each have a separate function • T-loop: recognition by the ribosome. • D loop: recognition by the aminoacyl tRNA synthetases • Anticodon loop: base pairs with the mRNA codon.

21. Coaxial stacking of stems • Base-paired stems often are involved in long-range interactions with other stems. • 7 bp acceptor stem in tRNA stacks on the 5 bp T stem to form an A-type helical arm. • The D stem and anticodon stem stack to form a second helical arm.

22. Common tertiary structure motifs in RNA • Many biological functions of RNA are based on very specific 3-D structures. • Interactions between preformed secondary structural domains.

23. Preformed secondary structural domains interact to form the tertiary structure. • RNA folding involves: Charge neutralization. Hydrogen bonding and base stacking. Noncanonical base pairing between distant nucleotides. Interactions between 2′-OH groups.

24. Pseudoknot motif • A single-stranded loop base pairs with a complementary sequence outside this loop. • Folds into a 3-D structure by coaxial stacking.

25. Telomerase RNA pseudoknot • Telomerase RNA provides the RNA template for telomere synthesis (see Chapter 6). • A highly conserved pseudoknot is required for telomerase activity. • Stabilized by base triple interactions.

26. A-minor motif • One of the most abundant long-range interactions in large RNA molecules. • Adenosines make contacts with the minor grooves of RNA helices.

27. Tetraloop motif • A stem-loop that is stabilized by base-stacking interactions. • e.g. the tetraloop sequence UUUU • Often include “G turns” • Hydrogen bond forms between the backbone phosphate and the 1-nitrogen of guanine.

28. Ribose zipper motif • Helix-helix interactions. • Hydrogen bonding between the 2'-OH of a ribose in one helix and the 2-oxygen of a pyrimidine base of the other helix.

29. Kink-turn motif • Asymmetric internal loops embedded in an RNA double helix. • Sharp bend (“kink”) in the phosphodiester backbone of a 3-nucleotide bulge.

30. Kissing hairpin loop • Two hairpin loops form a “kissing interaction” by base pairing between their single-stranded regions. • A “minor groove” is formed by the pseudo-continuous backbones of the hairpins.

31. Kinetics of RNA folding The “RNA folding problem” • Large RNAs are composed of a number of modular domains that assemble and fold independently. • There are many possible structures that a particular RNA chain can adopt. • Misfolding into a nonfunctional structure can occur due to incorrect base pairing.

32. Protein-mediated RNA folding • RNA chaperones either prevent the formation of misfolded structures or unfold misfolded RNA. • Specific RNA-binding proteins stabilize the native structure.

33. Kinetic folding profiles • Analyze by hydroxyl radical footprinting assay • tRNA Secondary structure within 10-4 to 10-5 seconds. Tertiary structure within 10-2 to 10-1 seconds. • Large catalytic RNA Catalytic center requires several minutes to complete folding.

34. 3.4 The discovery of RNA catalysis

35. Thousands of different chemical reactions are required to carry out essential processes in living cells. • Catalysis is necessary for biochemical reactions to proceed at a useful rate. • Catalysts lower activation energies.

36. Until the early 1980s it was assumed that biological catalysis depended exclusively on protein enzymes. • 1982: catalytic RNA discovered in a Tetrahymena ribosomal RNA intron. • 1983:RNA component of E. coli RNase P shown to be catalytic.

37. Tetrahymena self-splicing RNA • Thomas Cech and co-workers were studying transcription of ribosomal RNA (rRNA) genes in Tetrahymena thermophila. • Used the “R looping” technique: An RNA-DNA hybrid displaces one strand of duplex DNA. • Revealed an intervening sequence (IVS) or “intron” that is spliced out in the final 26S rRNA product.

38. Thomas Cech and colleagues developed an in vitro assay to study intron splicing. • In the presence of GTP and Mg2+ alone, the protein-free RNA underwent splicing. • Conclusion: the RNA was splicing itself.

39. Classic definition of an enzyme: A substance that increases the rate, or velocity, of a chemical reaction without itself being changed in the overall process. • Because many naturally occurring RNA enzymes are self-splicing, the term ribozyme was coined.

40. RNase P is a ribozyme • 1983: Sidney Altman and coworkers showed that bacterial RNase P is an RNA enzyme. • In vitro the M1 RNA alone can process precursor tRNA. • In vivo the C5 protein is required to enhance M1 RNA efficiency.

41. Eukaryotic RNase P acts as a catalytic ribonucleoprotein (RNP) • Human H1 RNA associates with  10 protein subunits. • H1 RNA plus two subunits (Rpp21 and Rpp29) are required for catalytic activity.

42. The RNA World • A hypothetical stage in the evolution of life when RNA both carried the genetic information and catalyzed its own replication.

43. Evidence for the RNA world hypothesis • RNA has all the structural prerequisites for self-replication. • RNA genomes are widespread among viruses. • RNA molecules: Self-fold into 3-D structures. Recognize other macromolecules and ligands with precision. Catalyze a diversity of reactions. • The ribosome is a ribozyme.

44. The RNP World • During a hypothetical transitional period, RNA catalyzed the synthesis of proteins and these proteins catalyzed the transition from RNA to DNA.

45. The DNA/RNA/Protein World • Today, proteins and ribonucleoprotein (RNP) particles catalyze: The replication of DNA. Transcription of DNA into RNA. Reverse transcription of RNA into DNA. • Translation of mRNA is mediated by the ribosome, a large ribozyme.

46. Ribozymes catalyze a variety of chemical reactions • Form substrate-binding sites. • Lower the activation energy. • Allow reaction to proceed much faster. • Many are metalloenzymes. e.g. binding of Mg2+ in active site

47. Mode of ribozyme action • Some ribozymes use a two-metal-ion mechanism for catalysis. • The active site of a self-splicing group I intron has the same orientation of two metal ions as found in a protein-based DNA polymerase. • Other ribozymes use general acid-base chemistry.

48. Naturally occurring ribozymes Two different groups based on difference in size and reaction mechanism: • Large ribozymes • Small ribozymes

49. Large ribozymes • Vary in size from a few hundred to 3000 nucleotides. • Cleave RNA to generate 3′-OH termini. • RNA components of RNase P, group I and group II intron family, RNA components of the spliceosome, ribosomal RNA.

50. Small ribozymes • Vary in size from 40 to 154 nucleotides. • Cleave RNA to generate a 2′-3′-cyclic phosphate and a product with a 5′-OH terminus. • Hammerhead motif, hairpin motif, hepatitis delta virus (HDV) RNA, Varkud satellite (VS) RNA, and the glmS riboswitch ribozyme. • Most are involved in self-replication. • Potential tools to combat viral disease.