Chapter 3:

The Versatility

of RNA

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

3.1 Introduction

• 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.

3.2 RNA is involved in a wide range of cellular processes

• Ribosomal RNA (rRNA)

• Messenger RNA (mRNA)

• Transfer RNA (tRNA)

• Small nuclear RNA (snRNA)

• Small nucleolar RNA (snoRNA)

• MicroRNA (miRNA)

Six major types of RNA

• RNA can form complementary base pairs with other nucleic acids.

• RNA can interact with proteins:

Ribonucleoprotein (RNP) particles

Two key points for understanding RNA function

• 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).

Overview of the versatility of RNA

3.3 Structural motifs of RNA

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).

• Secondary structure: the folding of an RNA chain into a variety of structural motifs.

• Common secondary structures can be predicted by computer analysis.

• 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.

Base-paired RNA adopts an A-type double helix

• >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

RNA helices often contain noncanonical base pairs

Base triples

• Typically involve one standard base pair.

• The third base interacts in a variety of unconventional ways.

RNA structures often contain unconventional base pairing

Important mediators of:

• RNA self-assembly.

• RNA-protein interactions.

• RNA-ligand interactions.

Noncanonical base pairs and base triples

“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.

General principles

• Modified bases.

• tRNA loops each have a separate function.

• Coaxial stacking of stems.

tRNA structure: important insights into RNA structural motifs

• 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

• 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.

Modified bases

• T-loop: recognition by the ribosome.

• D loop: recognition by the aminoacyl tRNA synthetases

• Anticodon loop: base pairs with the mRNA codon.

tRNA loops each have a separate function

• 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.

Coaxial stacking of stems

• Many biological functions of RNA are based on very specific 3-D structures.

• Interactions between preformed secondary structural domains.

Common tertiary structure motifs in RNA

• 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.

• A single-stranded loop base pairs with a complementary sequence outside this loop.

• Folds into a 3-D structure by coaxial stacking.

Pseudoknot motif

• 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.

Telomerase RNA pseudoknot

• One of the most abundant long-range interactions in large RNA molecules.

• Adenosines make contacts with the minor grooves of RNA helices.

A-minor 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.

Tetraloop 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.

Ribose zipper motif

• Asymmetric internal loops embedded in an RNA double helix.

• Sharp bend (“kink”) in the phosphodiester backbone of a 3-nucleotide bulge.

Kink-turn motif

• 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.

Kissing hairpin loop

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.

Kinetics of RNA folding

• RNA chaperones either prevent the formation of misfolded structures or unfold misfolded RNA.

• Specific RNA-binding proteins stabilize the native structure.

Protein-mediated RNA folding

• 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.

Kinetic folding profiles

3.4 The discovery of RNA catalysis

• 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.

• 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.

• 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.

Tetrahymena self-splicing RNA

• 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.

• 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.

• 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.

RNase P is a ribozyme

• Human H1 RNA associates with 10 protein subunits.

• H1 RNA plus two subunits (Rpp21 and Rpp29) are required for catalytic activity.

Eukaryotic RNase P acts as a catalytic ribonucleoprotein (RNP)

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

The RNA World

• 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.

Evidence for the RNA world hypothesis

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

The RNP 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.

The DNA/RNA/Protein World

• 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

Ribozymes catalyze a variety of chemical reactions

• 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.

Mode of ribozyme action

Two different groups based on difference in size and reaction mechanism:

• Large ribozymes

• Small ribozymes

Naturally occurring 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.

Large 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.

Small ribozymes

• The most frequently found catalytic motif in plant pathogenic RNAs.

• So called for its three helices in a T shape.

• The self-cleavage reaction proceeds by “in line” nucleophilic substitution.

• The 2′-OH of the ribose next to the phosphodiester bond to be cleaved acts as the attacking nucleophile.

Hammerhead motif

3.5 RNA-based genomes

• Mutation rate 1000-fold higher than DNA-based genomes.

• Lack of proofreading activity by RNA polymerases.

• Fitness trade-off between replication rate and replication fidelity.

• Several well-known viral diseases are due to RNA viruses.

Eukaryotic RNA viruses

• Infect many different hosts, including plants and animals.

• Many significant human or veterinary pathogens.

• Single-stranded or double-stranded RNA genome.

• Replicate without forming DNA intermediates.

• Enveloped or nonenveloped capsids.

Three main categories of eukaryotic RNA viruses

• Plus-strand (coding strand) virusese.g. agents of foot and mouth disease, common cold, polio

• Minus-strand (non-coding strand) virusese.g. agents of rabies, mumps, measles, influenza,

severe acute respiratory syndrome (SARS), fatal hemorrhagic fever

• Double-stranded RNAe.g. rotavirus, agent of infant diarrhea

Avian flu and swine flu

• Pandemics arise when a virus to which most people have no immunity, usually an avian strain, acquires the ability to transmit from animal to person…

….and then from person to person.

• The genes for two outer coat proteins, hemagglutinin and neuraminidase are constantly mutating.

• Possibility for exchange of genes with a common human flu strain.

• Reassortment produces new viruses with a variety of segment combinations.

Retroviruses

• RNA tumor viruses.

• Single-stranded RNA genome.

• Replicate through a DNA intermediate.

• Many well-known animal pathogens.

• One prominent human pathogen:

Human immunodeficiency virus 1 (HIV-1)

• Important vectors for gene therapy.

Viroids and other subviral pathogens

• Viroids are plant pathogens.

e.g. avocado sunblotch viroid and coconut cadang cadang viroid

• The viroid genome is a single, very small (250 to 400 nt), circular molecule of RNA.

• The viroid RNA is the infectious agent; it does not encode any protein.

• Viroids replicate using host-encoded RNA polymerase.

Satellite RNAs

• Single-stranded RNA genome.

• Found in plants and animals.

e.g. hepatitis delta virus, a small single-stranded RNA satellite of hepatitis B virus

• Some larger RNAs may encode a protein.

• Require a helper virus to replicate.

Virusoids

• Circular, single-stranded RNA genome.

• Plant pathogens that do not encode any protein.

e.g. velvet tobacco mottle, subterranean clover mottle

• Require a helper virus for replication and capsid formation.