Alan Weiner
BIOCHEM 530
Wednesday, EE 037
October 8, 2014
RNA structure and function
If RNA can be both
catalyst and genome,
RNA could replicate itself…
the first "living" molecule!
catalytic
(+) strand
template
(–) strand
precursors
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 RNApolymerase 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.
catalytic (+) strand copies
unfolded template (–) strand
two catalytic (+) strands
and original template (–) strand
Nucleic acids are plausible prebiotic condensation products
Nucleic acids are plausible prebiotic condensation products
generated by repeated dehydrations
=
O

5'
P

P
HN

P
uracil
O
N
O
O
OH OH
– H2O, glycosidic bond
ribose
– H2O, phosphoester bond (ribose,  phosphate)
– H2O, phosphoanhydride bonds (, and ,)
Whoops, right impulse, wrong concept!
What modern salvage pathways and "prebiotic" chemists do…
What Nature probably did…
Szostak (2009) Origins of life: Systems chemistry on
early Earth. Nature 459, 171-172 [News and Views]
"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
small
molecules
old-fashioned
chemical intuition
new-fangled
systems pathway
cyclic
intermediates
hydrolysis or
phosphorolysis
products
Powner et al. (2009) Nature 459, 239-242
The RNA World
Primordial Soup
Prebiotic World
• condensation of sugars,
bases, phosphate, and
random polynucleotides
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.
RNA World
• RNA genomes
• RNA enzymes
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!
RNP World
• protein synthesis
• protein enzymes
Progenote
DNA World
• DNA genomes
mt
chl
Eukaryotes
Eubacteria Archaea
Nucleotide cofactors could be molecular fossils
of metabolism in an RNA World
NH2
CH3
N
O
+
S
–O
N
N
N
O
+NH3
CH3
CH3
HO OH
S-adenosylmethionine (SAM)
OH
N
guanine
N
riboflavin-5'-phosphate
flavin mononucleotide (FMN)
N
N
NH2
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
5'
PfrA coding region
3'
a virulence-activating
transcription factor
in Listeria monocytogenes
RNA secondary structure
in 5’ untranslated region
(5’ UTR) is a thermosensor:
stable at 30°C, melts at 37°C
[Johansson et al. (2002)
Cell 110, 551–561]
"aptamer"
5'
coding region
3'
proteins involved in methyl metabolism (SAM),
purine metabolism and transport (guanine),
and flavin mononucleotide biosynthesis (FMN)
in Bacillus subtilis
RNA secondary structures
can 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]
Breaker (2004) Nature 432, 838;
(2007) Nature 447, 497
If RNA can be both
catalyst and genome,
RNA could replicate itself…
the first "living" molecule!
catalytic
(+) strand
template
(–) strand
precursors
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 RNApolymerase 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.
catalytic (+) strand copies
unfolded template (–) strand
two catalytic (+) strands
and original template (–) strand
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 tree
stunted by exocortis
http://www.dpi.nsw.gov.au/agriculture/horticulture/citrus/health/diseases/citrus-exocortis
Hammerhead ribozyme structure
hydrolysis
cartoon showing stems, loops,
and 17 conserved nucleotides
backbone trace
of actual structure
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
attacking
R3-OH
O
before
tetrahedral phosphate
with two ester bonds
(phosphodiester)
P
If R3-OH is RNA-OH,
the result is
RNA isomerization.
If R3-OH is H-OH
(water), the result is
RNA hydrolysis.
R1-O
H
R3-O
O–
O-R2
O
P
O-R2
O–
during
attacking hydroxyl
generates a trigonal,
bipyramidal intermediate
R1-O
O
P
R3-O
O–
O-R1
R2-OH
expelled
after
bipyramidal intermediate
turns "inside out"
like umbrella in high wind
("inversion of configuration")
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
O
– =
H 2O
O-P-O
O
O
– =
If the attacking hydroxyl
belongs to H2O, the RNA
is hydrolyzed (cleaved).
OH + HO-P-O
O
– =
If the attacking hydroxyl
belongs to RNA, the
RNAs are isomerized.
O
O-P-O
O
O
– =
OH
O-P-O
O +
OH
RNA splicing and replication are chemically (almost) identical
OH
– =
O-P-O
O
O
– =
O-P-O
O +
O
O
O-P-O -P-O -P-OO O O
O-P-OO
O
NOH
– =
– =
O
NOH
O
– =
– =
If the RNA 3' hydroxyl attacks a
ribonucleotide triphosphate (NTP),
the reaction is a polymerization
as in replication!
OH
– =
OH
O
– =
If the attacking hydroxyl
belongs to RNA,
the RNAs are isomerized
as in splicing.
O
+ O -P-O -P-OO O
mRNA splicing is just an isomerization…
a new bond is made for every bond broken
2’
3’
3’
2’, 3’, 5’
The smallest natural ribozymes are the hepatitis  ribozyme
and the hammerhead ribozyme found in plant viroids
3'
2'
The "hairpin" ribozyme cleaves
the negative strand of tobacco
ringspot virus satellite RNA
5'
•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
The transition state is stabilized
by 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
Transition State Stabilization by a Catalytic RNA
(Rupert, Massey, Sigurdsson, and Ferré-D'Amaré, 2002)
Precursor and product differ "infinitesimally" from the transition state…
precursor
+ vanadate model
precursor
product
+ vanadate model
product
The smallest natural ribozymes are the hepatitis  ribozyme
and the hammerhead ribozyme found in plant viroids
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!
Doudna and Lorsch (2005)
Nat Struct Mol Biol 12, 395-402.
mRNA life history is regulated at every step
from synthesis to destruction
cytoplasm
nucleus
5'
unspliced
mRNA
DNA
RNA polymerase
spliced
mRNA
5' cap
AAAA 3'
Flavors of alternative mRNA splicing
default splicing pattern
5’ cap
1
2
3
4
AAAAAAAAA 3’
exon skipping or exclusion (+/– exon 2)
5’ cap
1
2
3
4
alternative exons (include exon 2 or 3)
AAAAAAAAA 3’
Current world record holder for alternative mRNA splicing is
the Drosophila DSCAM gene, whose protein products
function in axon guidance and innate immunity in the fly
exon 4
exon 6
12 alternatives 48 alternatives
exon 9
33 alternatives
exon 17
2 alternatives
genomic DNA
and
pre-mRNA
mRNA
5'
protein
N
3'
C
outside
TM
inside
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
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...
outside
Hattori et al. (2007) Dscam
diversity is essential for
neuronal wiring and selfrecognition. 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.
.
inside
Current world record holder for alternative mRNA splicing is
the Drosophila DSCAM gene, whose protein products
function in axon guidance and innate immunity in the fly
exon 4
exon 6
12 alternatives 48 alternatives
exon 9
33 alternatives
exon 17
2 alternatives
genomic DNA
and
pre-mRNA
mRNA
5'
protein
N
3'
C
outside
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 pathogens
TM
inside
Schmucker 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.
18
Gilbert, 1986
18
18
18
17
18
17
36
18
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.
18
17
18
17
X
18
17
18
17
18
17
18
17
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
U4
5’ exon
U1 U5 U6
U2
3’ exon
U snRNPs assemble
into a spliceosome
on mRNA precursor
3’ exon
first catalytic
step generates
an RNA lariat
U1 U4
U5 U6
U2
5’ exon
3’ exon
+
U5 U6
U2
second catalytic step
ligates exons and
releases lariat intron
with U snRNPs
The five spliceosomal snRNAs (major class)
-
5’ splice site
recognition
U5
branch site
recognition
UC
U G
C-G
G-C
U
U-A
C-G
G-C
U-A
G-C
CH3Oppp
AUAUACUAAAAUUGGAACGAUACAGA
A
U
A
U
C
U
G
A
U
A
AG UCCUCU
A
U
catalysis
U2
GACACGCAAAUUCGUGAAGCGUUCCAUA
U
A UU
G- ApppG 2,2,7 m3
GU
CG
A
U
U
G
AA
U
C
A
G - U
C- - G
G
C
GG-C AGUUUUUAA
CCCCAUAACCCUUUUCAAAAG U - A
GC-G
U
G
U -U
C C- - C
U-A
G-C
A -U
A
C
A -A
C-G
G-C
C - G
U -G
U
G G- - G U
A-U
U-G
C
C G
G-C
G -A U- C
A-U
GA
U
A-U
C-G
U
G
A
A UU-A U
G-C
G-C
G U
A-U
CA
C-G
G-C
U-A
C-G
G-C
G-U
A
A-U
C-G
U U
G-C
A
G
C
U
A G-C A
AA
G
A
C
U
C
G
U
A
A
U U U GG
U6
OH
UUU
C
U
C
A
G
C
C
U
U–A
U–A
U–A
C–G
U–A
A–U
A–Y
AU A – U
C
C
G
C
C
G
UA
GA – U
C–G
U–G
U–A
C–G
U–A
C–G
U–G
U–A
UU
C
G
U–A
C–G
G – C AA
G–C
G
C
U–A
U
U
U–A
C – G UC
C–G
U–A
C–G
C–G
G–C
2,2,7m GpppAUA – UCUUAACCCAAUUUUUUGA – UA
3
OH
A
U U
U
U
U
U
C-G
C-G
U
GG-C
AG-U
CC
A
U-A
UC-G
AU-A
Um 2,2,7
G
ppp
AUCGCU
UCAAGUGUAGUAUCUGUUCU
3
U U
C
G
G- C
A- U
G- C
G- C
A
U
G
A
A- U
G- C
G- C
U-A
A
G- C
A- U
G- C
UU
G
A
G- C
A
C
AAUAUAUUAAAUGGAUUUUUGGAGCAG
C
U
C
GCAUCGACCUGG
A
A
CGUGGC GGACC
G
G
A
U
AA
U
C
CC
G
A
CA
A
OH
G
C
C
OH
-
U1
catalysis
-
GC A C
U
U
C
U
A
C
C-G
C-G
U-A
A-U
U-G
U
U
C-G
G-C
G-U
A G
UC
G
A
U G
C C
G-C
G-C
G-C
C-G
G-C
G-C
A
A-U
U
U
A
A
A G
C
G
G
C-G
U
C
A
CGA UUUCCC
UGGU UUUCC
C
A-U
U
A
GCU AAAGGG G
ACCA AGAGG
G-C
G A
C
U
C
UU
U
G-C
U AG
G-C
G-C
A-U
C-G
G-C
G-C
G-UOH
m 2,2,7
G
AUACUUACCUG
ppp
AUAAUUUGUGGUAGU
3
U4
repressor
and
(re)assembly
factor
The five spliceosomal snRNAs (major class)
-
5’ splice site Sm site
recognition
U5
Sm site
branch site
recognition
UC
U G
C-G
G-C
U
U-A
C-G
G-C
U-A
G-C
CH3Oppp
AUAUACUAAAAUUGGAACGAUACAGA
A
U
A
U
C
U
G
A
U
A
AG UCCUCU
A
U
catalysis
U2
Sm site
GACACGCAAAUUCGUGAAGCGUUCCAUA
U
A UU
G- ApppG 2,2,7 m3
GU
CG
A
U
U
G
AA
U
C
A
G - U
C- - G
G
C
GG-C AGUUUUUAA
CCCCAUAACCCUUUUCAAAAG U - A
GC-G
U
G
U -U
C C- - C
U-A
G-C
A -U
A
C
A -A
C-G
G-C
C - G
U -G
U
G G- - G U
A-U
U-G
C
C G
G-C
G -A U- C
A-U
GA
U
A-U
C-G
U
G
A
A UU-A U
G-C
G-C
G U
A-U
CA
C-G
G-C
U-A
C-G
G-C
G-U
A
A-U
C-G
U U
G-C
A
G
C
U
A G-C A
AA
G
A
C
U
C
G
U
A
A
U U U GG
OH
UUU
C
U
C
A
G
C
C
U
U–A
U–A
U–A
C–G
U–A
A–U
A–Y
AU A – U
C
C
G
C
C
G
UA
GA – U
C–G
U–G
U–A
C–G
U–A
C–G
U–G
U–A
UU
C
G
U–A
C–G
G – C AA
G–C
G
C
U–A
U
U
U–A
C – G UC
C–G
U–A
C–G
C–G
G–C
2,2,7m GpppAUA – UCUUAACCCAAUUUUUUGA – UA
3
OH
A
U U
U
U
U
U
C-G
C-G
U
GG-C
AG-U
CC
A
U-A
UC-G
AU-A
Um 2,2,7
G
ppp
AUCGCU
UCAAGUGUAGUAUCUGUUCU
3
U U
C
G
G- C
A- U
G- C
G- C
A
U
G
A
A- U
G- C
G- C
U-A
A
G- C
A- U
G- C
UU
G
A
G- C
A
C
AAUAUAUUAAAUGGAUUUUUGGAGCAG
C
U
C
GCAUCGACCUGG
A
A
CGUGGC GGACC
G
G
A
U
AA
U
C
CC
G
A
CA
A
OH
G
C
C
Sm site
U6
OH
-
U1
catalysis
-
GC A C
U
U
C
U
A
C
C-G
C-G
U-A
A-U
U-G
U
U
C-G
G-C
G-U
A G
UC
G
A
U G
C C
G-C
G-C
G-C
C-G
G-C
G-C
A
A-U
U
U
A
A
A G
C
G
G
C-G
U
C
A
CGA UUUCCC
UGGU UUUCC
C
A-U
U
A
GCU AAAGGG G
ACCA AGAGG
G-C
G A
C
U
C
UU
U
G-C
U AG
G-C
G-C
A-U
C-G
G-C
G-C
G-UOH
m 2,2,7
G
AUACUUACCUG
ppp
AUAAUUUGUGGUAGU
3
U4
repressor
and
(re)assembly
factor
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 snRNPs
recognize the 5’ and 3’ splice junctions
and catalyze the mRNA splicing reaction
2’
OH
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
GroupII
Group
(in cis)
GroupIIII
Group
(in cis)
U2/U6
U1
?
2
U5
1
U2/branch site
2
1
mRNA
mRNA
(major
class)
(in trans)
-
5’ splice site
recognition
U5
branch site
recognition
UC
U G
C-G
G-C
U
U-A
C-G
G-C
U-A
G-C
CH3Oppp
AUAUACUAAAAUUGGAACGAUACAGA
A
U
A
U
C
U
G
A
U
A
AG UCCUCU
A
U
U2
catalysis
GACACGCAAAUUCGUGAAGCGUUCCAUA
U
A UU
G- ApppG 2,2,7 m3
GU
CG
A
U
U
G
AA
U
C
A
G - U
C- - G
G
C
GG-C AGUUUUUAA
CCCCAUAACCCUUUUCAAAAG U - A
GC-G
U
G
U -U
C C- - C
U-A
G-C
A -U
A
C
A -A
C-G
G-C
C - G
U -G
U
G G- - G U
A-U
U-G
C
C G
G-C
G -A U- C
A-U
GA
U
A-U
C-G
U
G
A
A UU-A U
G-C
G-C
G U
A-U
CA
C-G
G-C
U-A
C-G
G-C
G-U
A
A-U
C-G
U U
G-C
A
G
C
U
A G-C A
AA
G
A
C
U
C
G
U
A
A
U U U GG
U6
OH
UUU
C
U
C
A
G
C
C
U
U–A
U–A
U–A
C–G
U–A
A–U
A–Y
AU A – U
C
C
G
C
C
G
UA
GA – U
C–G
U–G
U–A
C–G
U–A
C–G
U–G
U–A
UU
C
G
U–A
C–G
G – C AA
G–C
G
C
U–A
U
U
U–A
C – G UC
C–G
U–A
C–G
C–G
G–C
2,2,7m GpppAUA – UCUUAACCCAAUUUUUUGA – UA
3
OH
A
U U
U
U
U
U
C-G
C-G
U
GG-C
AG-U
CC
A
U-A
UC-G
AU-A
Um 2,2,7
G
ppp
AUCGCU
UCAAGUGUAGUAUCUGUUCU
3
U U
C
G
G- C
A- U
G- C
G- C
A
U
G
A
A- U
G- C
G- C
U-A
A
G- C
A- U
G- C
UU
G
A
G- C
A
C
AAUAUAUUAAAUGGAUUUUUGGAGCAG
C
U
C
GCAUCGACCUGG
A
A
CGUGGC GGACC
G
G
A
U
AA
U
C
CC
G
A
CA
A
OH
G
C
C
OH
-
U1
catalysis
-
GC A C
U
U
C
U
A
C
C-G
C-G
U-A
A-U
U-G
U
U
C-G
G-C
G-U
A G
UC
G
A
U G
C C
G-C
G-C
G-C
C-G
G-C
G-C
A
A-U
U
U
A
A
A G
C
G
G
C-G
U
C
A
CGA UUUCCC
UGGU UUUCC
C
A-U
U
A
GCU AAAGGG G
ACCA AGAGG
G-C
G A
C
U
C
UU
U
G-C
U AG
G-C
G-C
A-U
C-G
G-C
G-C
G-UOH
m 2,2,7
G
AUACUUACCUG
ppp
AUAAUUUGUGGUAGU
3
Are the five spliceosomal snRNAs really
fragments of a Group II self-splicing intron?
U4
repressor
and
(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 RNA
protein makes offer
RNA cannot refuse
RNA relaxes, now
protein-dependent