Alan Weiner BIOCHEM 530 Wednesday, EE 037 October 8, 2014 RNA structure and function

Alan WeinerBIOCHEM 530Wednesday, EE 037October 8, 2014RNA structure and function

If RNA can be bothcatalyst and genome,

RNA could replicate itself… the first "living" molecule!

catalytic(+) strand

template(–) strand

catalytic (+) strand copies unfolded template (–) strand

two catalytic (+) strandsand original template (–) strand

Lawrence and Bartel (2005) New ligase-derived RNA polymerase ribozymes. RNA 11, 1173-1180; Shechner and Bartel (2011) The structural basis of RNA-catalyzed RNA polymerization. Nat Struct Mol Biol 18, 1036-1042; Shechner et al. (2009) Crystal structure of the catalytic core of an RNA-polymerase ribozyme. Science 326, 1271-1275; Bagby et al. (2009) A class I ligase ribozyme with reduced Mg2+ dependence: Selection, sequence analysis, and identification of functional tertiary interactions. RNA 15, 2129-2146.

precursors

Nucleic acids are plausible prebiotic condensation products

Nucleic acids are plausible prebiotic condensation products generated by repeated dehydrations

uracil

O

OH OH

P O

O

=

O =

5' – H2O, glycosidic bond

– H2O, phosphoester bond (ribose, phosphate)

ribose

PP

– H2O, phosphoanhydride bonds (, and ,)

HN

N

Whoops, right impulse, wrong concept!

Szostak (2009) Origins of life: Systems chemistry on early Earth. Nature 459, 171-172 [News and Views]

What Nature probably did…

What modern salvage pathways and "prebiotic" chemists do…

"Systems chemistry on early earth"… proposed by Oparin in 1938, tested by Miller in 1953 as his PhD Thesis!

"Systems chemistry on early earth"…Options for pyrimidine ribonucleotide assembly

Powner et al. (2009) Nature 459, 239-242

smallmolecules

cyclic intermediates hydrolysis or

phosphorolysisproducts

old-fashionedchemical intuition

new-fangledsystems pathway

The RNA World

If RNA is catalytic, it could function as both genome and replicase, replicating itself, and perhaps also encoding ribozymes that would carry out intermediary metabolism to make more RNA precursors.

We can debate whether an RNA World existed, or how complex it might have been, and whether RNA may have been preceded by a simpler RNA-like polymer that could also function as genome and replicase.

However, if life did begin in an RNA-like World, it may still be an RNA World today only slightly disguised by a veil of DNA!

Prebiotic World• condensation of sugars, bases, phosphate, and random polynucleotides

Primordial Soup

Progenote

Eukaryotes Eubacteria Archaea

mtchl

DNA World• DNA genomes

RNA World• RNA genomes• RNA enzymes

RNP World• protein synthesis• protein enzymes

S-adenosylmethionine (SAM)

riboflavin-5'-phosphateflavin mononucleotide (FMN)

Nucleotide cofactors could be molecular fossils of metabolism in an RNA World

guanine

CH3

CH3

NH2

OH

N

NN

N

CH3

NH2

N

NN

NSO

HO OH

+NH3

O

–O

+

White HB (1976) Coenzymes as fossils of an earlier metabolic state. J Mol Evol 7, 101-104

Riboswitches can regulate mRNA expression without help from proteins or transcription

a virulence-activating transcription factor

in Listeria monocytogenes

PfrA coding region

RNA secondary structurein 5’ untranslated region

(5’ UTR) is a thermosensor:stable at 30°C, melts at 37°C

[Johansson et al. (2002)Cell 110, 551–561]

proteins involved in methyl metabolism (SAM), purine metabolism and transport (guanine),

and flavin mononucleotide biosynthesis (FMN)in Bacillus subtilis

coding region

RNA secondary structurescan bind small metabolites

such as SAM, guanine, and FMN,coordinately controlling genes

involved in methyl, purine,and cofactor metabolism

[Winkler et al. (2003)Nat Struct Biol 10, 701–707]

5' 3'

5' 3'

Breaker (2004) Nature 432, 838; (2007) Nature 447, 497

"aptamer"

If RNA can be bothcatalyst and genome,

RNA could replicate itself… the first "living" molecule!

catalytic(+) strand

template(–) strand

catalytic (+) strand copies unfolded template (–) strand

two catalytic (+) strandsand original template (–) strand

Lawrence and Bartel (2005) New ligase-derived RNA polymerase ribozymes. RNA 11, 1173-1180; Shechner and Bartel (2011) The structural basis of RNA-catalyzed RNA polymerization. Nat Struct Mol Biol 18, 1036-1042; Shechner et al. (2009) Crystal structure of the catalytic core of an RNA-polymerase ribozyme. Science 326, 1271-1275; Bagby et al. (2009) A class I ligase ribozyme with reduced Mg2+ dependence: Selection, sequence analysis, and identification of functional tertiary interactions. RNA 15, 2129-2146.

precursors

The smallest natural ribozymes are the hepatitis ribozyme and the hammerhead ribozyme found in plant viroids

Australia battled citrus exocortis virus (oranges, lemons, and etrogs on susceptible rootstocks) in the 1940s and

1950s

healthy treestunted by exocortis

http://www.dpi.nsw.gov.au/agriculture/horticulture/citrus/health/diseases/citrus-exocortis

Hammerhead ribozyme structure

cartoon showing stems, loops, and 17 conserved nucleotides

backbone traceof actual structure

hydrolysis

Martick et al. (2006) A discontinuous hammerhead ribozyme embedded in a mammalian [lectin] messenger RNA. Nature 454, 899-902; Chi et al. (2008) Capturing hammerhead ribozyme structures in action by modulating general base catalysis. PLoS Biol 6, e234.

All natural ribozymes (except the ribosome!) use the same mechanism:the attacking hydroxyl group expels the leaving hydroxyl group,

making a new phosphoester bond as it breaks the old one

If R3-OH is RNA-OH, the result is

RNA isomerization.

If R3-OH is H-OH (water), the result is

RNA hydrolysis.

beforetetrahedral phosphatewith two ester bonds(phosphodiester)

duringattacking hydroxylgenerates a trigonal,bipyramidal intermediate

afterbipyramidal intermediateturns "inside out"like umbrella in high wind("inversion of configuration")

P

O-R2

O–

O

R1-O

attacking

R3-OH

P

O-R1

O–

O

R3-O

R2-OHexpelled

P

O–

O

R3-O O-R2

R1-O

H

All natural ribozymes (except the ribosome!) use the same mechanism:the attacking hydroxyl group expels the leaving hydroxyl group,

making a new phosphoester bond as it breaks the old one

If the attacking hydroxyl belongs to H2O, the RNA is hydrolyzed (cleaved).

If the attacking hydroxyl belongs to RNA, the

RNAs are isomerized.

H2O

O-P-O

O

O

=–

HO-P-O

O

OH

=–

O

+

OH

O-P-O

O

O

=–

OH+

O-P-O

O

=–

O

If the attacking hydroxyl belongs to RNA,

the RNAs are isomerizedas in splicing.

OH

O-P-O

O

O

=–

OH+

O-P-O

O

=–

O

If the RNA 3' hydroxyl attacks aribonucleotide triphosphate (NTP),

the reaction is a polymerizationas in replication!

OH

O-P-O

O

O

=–

+O-P-O-

O=

–

O

-P-O

O

O

=– -P-O-

O

O

=– N

N -P-O

O

O

=– -P-O-

O

O

=–O

RNA splicing and replication are chemically (almost) identical

OH

OH

mRNA splicing is just an isomerization…a new bond is made for every bond broken

2’

3’

3’

2’, 3’, 5’

The "hairpin" ribozyme cleavesthe negative strand of tobacco

ringspot virus satellite RNA

•To crystallize "the" precursor, the attacking 2'-OH was replaced by the methoxy group 2'-OCH3 which cannot dissociate.

•To crystallize "the" transition state, the scissile phosphate was deleted, and the ribozyme was complexed with vanadate (Na3VO4) to mimick the pentacovalent bipyramidal intermediate.

•The product crystallizes naturally (and apparently does not undergo the reverse reaction!).

Rupert, Massey, Sigurdsson, and Ferré-D'Amaré, 2002

2'3'

5'

The transition state is stabilizedby more hydrogen bonds

than either precursor or product

(A) Precursor complex stabilized by two hydrogen bonds (dashed lines) from G8.

(B) Transition state mimic complex stabilized by five hydrogen bonds. (The green triangle connects the equatorial oxygens of the vanadate.) Protonation is inferred from the distance between N1 of A38 and the 5'-oxygen.

(C) Product complex stabilized by three hydrogen bonds. The hydrogen bond between N1 of A38 and the 5'-OH of G+1 orients the latter for subsequent ligation.

Rupert, Massey, Sigurdsson, and Ferré-D'Amaré, 2002

precursor

product

precursor+ vanadate model

product+ vanadate model

Transition State Stabilization by a Catalytic RNA(Rupert, Massey, Sigurdsson, and Ferré-D'Amaré, 2002)

Precursor and product differ "infinitesimally" from the transition state…

Doudna and Lorsch (2005)Nat Struct Mol Biol 12, 395-402.

Doudna and Lorsch (2005)Nat Struct Mol Biol 12, 395-402.

Ribozyme-catalyzed reactions: very different,and often far better*

* think of ribosomes and spliceosomes which require large motions of large complexes with many RNA molecules

coming and going!

mRNA life history is regulated at every stepfrom synthesis to destruction

5'

RNA polymerase

DNA

unsplicedmRNA

nucleus

cytoplasm

spliced mRNA

5' cap AAAA 3'

Flavors of alternative mRNA splicing

1 2 3 45’ cap AAAAAAAAA 3’

default splicing pattern

exon skipping or exclusion (+/– exon 2)

1 2 3 45’ cap AAAAAAAAA 3’

alternative exons (include exon 2 or 3)

Current world record holder for alternative mRNA splicing isthe Drosophila DSCAM gene, whose protein products

function in axon guidance and innate immunity in the fly

The 61 kb DSCAM gene generates an 8 kb mRNA containing 24 exons. Exons 4, 6, 9, and 17 are encoded as tandem arrays of mutually exclusive alternative exons, so this one gene could in principle generate as many as 12 48 33 2 or 38,016 different

mRNAs and proteins. McManus and Graveley (2011) Curr Op Genet Dev 21, 373-379

exon 412 alternatives




protein N

genomic DNA and

pre-mRNA

TMoutside inside

3'

C

mRNA 5'

New answers pose new questions. We assumed that transcriptional control would be the major determinant of complexity, and we asked What wires transcription? Now it turns out that nerves are wired by alternative mRNA splicing, and so we must now ask What wires alternative splicing? Maybe not an endless hall of mirrors, but certainly no end in sight...

Hattori et al. (2007) Dscam diversity is essential for neuronal wiring and self-recognition. Nature 449, 223-228; Meijers et al. (2007) Structural basis of Dscam isoform specificity. Nature 449, 487-491; Du Pasquier (2005) Insects diversify one molecule to serve two systems. Science 309, 1826-1827..

insideoutside

Current world record holder for alternative mRNA splicing isthe Drosophila DSCAM gene, whose protein products

function in axon guidance and innate immunity in the fly





protein N

genomic DNA and

pre-mRNA

TMoutside inside

3'

C

mRNA 5'

Immunoglobulin folds with alternative regions (color) and constant regions (black):

homophilic interactions between similar DSCAM variants presumably play a role in

axon guidance; alternative regions presumably function in innate immunity

against microbial pathogensSchmucker et al. (2000) Drosophila Dscam Is an Axon Guidance Receptor Exhibiting Extraordinary Molecular Diversity. Cell 101, 671-684

The exon theory of genes: complex genes were assembled from small coding exons, and the

assembled exons were then joined together by mRNA splicing.

Accidental DNA recombinations occuring over evolutionary time are

random and sloppy…

but mRNA splicing is smart, and precisely joins each coding region

to the next.

Gilbert, 1986

17 17

17 17

17 17

17 17

X

18

18 18 1836

18

18 18

18 18

18

18 18

mRNA splicing is just an isomerization…a new phosphoester bond is made for each bond broken

2’

3’

3’

2’, 3’, 5’

U1, U2, U4, U5, and U6 small nuclear RNA (snRNAs)collaborate to splice out mRNA introns

second catalytic step ligates exons and releases lariat

intron with U snRNPs

U1

U2

U5 U6

U4

U2

U5 U6

U4

U snRNPs assemble into a spliceosome on

mRNA precursor

first catalytic step generates an RNA lariat

+U2

U5 U6

5’ exon 3’ exon

5’ exon

5’ exon

3’ exon

3’ exon

U1

U5

U1

The five spliceosomal snRNAs (major class)

U4

U6catalysi

s

repressorand

(re)assembly factor

branch siterecognition

U2

catalysis

5’ splice siterecognition

U5

U1

The five spliceosomal snRNAs (major class)

U4

U6


catalysis

repressorand

(re)assembly factor


U2

catalysis

Sm site

Sm site

Sm site

Sm site

Sm proteins form a heptameric ring, and are found in Archaea!

Kambach, Walke, Young, Avis, de la Fortelle, Raker, Lührmann, Li, and Nagai (1999) Cell 96, 375.

The snRNA components of the snRNPsrecognize the 5’ and 3’ splice junctions

and catalyze the mRNA splicing reaction

OH2’

U1 snRNA U2 snRNA

mRNA splicing is mechanistically identical to "Group II" autocatalytic self-splicing

2’

3’

3’

2’, 3’, 5’

Three kinds of RNA splicing

U2/branch site

U2/U6U1

Group I

Group II

mRNA(major class)

mRNA(in trans)

Group I(in cis)

Group II(in cis)

?

1

2

2

1U5

U5

U1

Are the five spliceosomal snRNAs really fragments of a Group II self-splicing intron?

U4

U6



catalysis

U2

catalysis

repressorand

(re)assembly factor

Group II introns in ancient protein coding genes could

have evolved in situ into modern mRNA introns by sequential replacement of

Group II domains with snRNAs working in trans

Jarrell, Dietrich, Perlman (1988)Mol Cell Biol 8, 2361-2366

How the RNP got its protein…an offer it could not refuse?

independent RNAprotein makes offer RNA cannot refuse

RNA relaxes, now protein-dependent

Alan Weiner BIOCHEM 530 Wednesday, EE 037 October 8, 2014 RNA structure and function

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