test

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Alan Weiner BIOC 530 December 7, 2007 RNA, and
why you should care about it Part III
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mRNA splicing is just an isomerizationa new
bond is made for every bond broken
2
3
3
2, 3, 5
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mRNA splicing is mechanistically identical to
"Group II" autocatalytic self-splicing
2
3
3
2, 3, 5
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U1, U2, U4, U5, and U6 small nuclear RNA (U
snRNAs)collaborate to splice out mRNA introns
U4
U1
U5
U6
U snRNPs assemble into a spliceosomeon mRNA
precursor
5 exon
3 exon
U2
U1
U4
U5
U6
first catalytic step generates an RNA lariat
3 exon
U2
second catalytic step ligates exons and releases
lariat intron with U snRNPs
U5
U6

5 exon
3 exon
U2
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The five spliceosomal snRNAs (major class)
U1
U2
U6
U5
U4
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The snRNA components of the U snRNPsrecognize
the 5 and 3 splice junctionsand catalyze the
mRNA splicing reaction
bulged branch site A attacks 5' splice site
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2'OH
OH
U1 snRNArecognizes5' splice site
U2 snRNArecognizesbranch site
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The five spliceosomal snRNAs (major class)
catalysis
U1
U2
branch site recognition
5 splice site recognition
U5
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Three kinds of RNA splicing
Group I (in cis)
Group I
Group II (in cis)
Group II
U2/U6
?
2
U2/branch site
1
2
mRNA (major class)
mRNA (in trans)
1
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mRNA and Group II splicing are closely related
and perhaps nearly identical
mRNA
Group II
Madhani and Guthrie (1992) Cell 71, 803817
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Splicing-related catalysis by protein-free U
snRNAs Valadkhan and Manley (2001) Nature 413,
695696
aberrant but apparently meaningful
reaction generates phosphotriester instead of
lariat
branch oligonucleotide
branch site A
Complex between U2, U6, and the branch
oligonucleotide assembled in vitro. Invariant
regions of U6 are shaded previously established
base-paired regions are indicated the branch
site consensus is underlined and a genetically
proved interaction in yeast is indicated by
arrowheads. Also indicated are psoralen
crosslinks (dashed boxes), UV crosslinks (circled
residues connected by zigzags), and the covalent
link that forms between the branch site and U6
snRNA in the protein-free in vitro reaction (24
hours, 0.1 conversion). Nucleotides are numbered
as in full length human U2 and U6 snRNA.
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Three kinds of RNA splicing
Group I (in cis)
Group I
Group II (in cis)
Group II
U2/U6
?
2
U2/branch site
1
2
mRNA (major class)
mRNA (in trans)
1
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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
inspired by, but not taken from, Jarrell,
Dietrich, Perlman (1988) Mol Cell Biol 8,
2361-2366
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How the RNP got its proteinan offer it could
not refuse?
happy,independent RNA
protein makes offer RNA cannot refuse
RNA relaxes, now protein-dependent
HB White, B Alberts
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Sm proteins form a heptameric ring
Kambach, Walke, Young, Avis, de la Fortelle,
Raker, Lührmann, Li, and Nagai (1999) Cell 96
375.
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Sm proteins are ancient proteins, also found in
Archaea, that bind oligo(U) or AUUUUUG. Sm
proteins may be more important for intracellular
trafficking or localization than for function.
UV
Urlaub, Raker, Kostka, and Lührmann (2001) EMBO J
20187 Achsel, Stark, and Lührmann (2001) PNAS
98 3685.
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Primordial Soup
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. 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
RNA World RNA genomes RNA enzymes
RNP World protein synthesis protein enzymes
DNA World DNA genomes
Progenote
mt
chl
Eukaryotes
Eubacteria
Archaea
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Primordial Soup
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. 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
RNA World RNA genomes RNA enzymes
RNP World protein synthesis protein enzymes
reverse transcriptase
DNA World retroelements DNA genomes
Progenote
mt
chl
Eukaryotes
Eubacteria
Archaea
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The human genome is chock full of parasitic
retrotransposable elements (retrotransposons)
that use a reverse transcriptase to replicateand
transpose through RNA intermediates
percentage of genome
structure of retroelement
copy number
autonomy
length
class
ORF1 RT-INT
LINEs
autonomous
850,000
21
6-8 kb
AAA
SINEs
non-autonomous
1,500,000
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100-300 bp
AAA
gag RT-INT env

autonomous
6-11 kb
retrovirus-like elements
450,000
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gag
non-autonomous
1.5-3 kb

autonomous
2-3 kb
DNA transposonfossils
300,000
3
non-autonomous
80-3,000 bp
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Retroviruses and retrotransposable elements made
(ridiculously) simple
RNA
retroviral particle
extracellular
intracellular
retroviral particle
RNA
translation of LINE RNA to generate reverse
transcriptase and integrase also used by
noncoding SINEs
RNA
RNA
translation to generate reverse transcriptase,
integrase, and capsid proteins for viral assembly
reverse transcription
reverse transcription
RNA
RNA
second strand synthesis
second strand synthesis
transcription
transcription
integration
integration
parental retroelement
daughter retroelement
provirus
genome
genome
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The human genome is chock full of parasitic
retrotransposable elements (retrotransposons)
that use a reverse transcriptase to replicateand
transpose through RNA intermediates
percentage of genome
structure of retroelement
copy number
autonomy
length
class
ORF1 RT-INT
LINEs
autonomous
850,000
21
6-8 kb
AAA
SINEs
non-autonomous
1,500,000
13
100-300 bp
AAA
gag RT-INT env

autonomous
6-11 kb
retrovirus-like elements
450,000
8
gag
non-autonomous
1.5-3 kb

autonomous
2-3 kb
DNA transposonfossils
300,000
3
non-autonomous
80-3,000 bp
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Caenorhabditis elegans (aka C. elegans, "the
worm", nematode, roundworm)
1 mm
Zamore and Haley (2005) Ribo-gnome The big world
of small RNAs. Science 309, 1519-1524
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Six easy steps to understanding RNAi
Step One. A perfect RNA duplex is usually a sign
of danger.
bidirectional transcription of a compact viral
genome
RNA
viral DNA genome
RNA
transcription of dispersed repeated retroelements
("genomic parasites")
Alu RNA
L1 RNA
gene
genomic DNA
Alu
L1
L1
Alu
Alu
Alu RNA
Alu RNA
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Six easy steps to understanding RNAi
Step Two. The RNA duplex is "diced" into 21 bp
fragments.
RNA
long RNAs
RNA
"dicer"-like enzymes
21 bp RNAs
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Six easy steps to understanding RNAi
Step Three. One RNA strand associates with a
RISC-like complex (RISC
RNA-induced silencing complex)
RNA
long duplex RNAs
RNA
"dicer"-like enzymes
21 bp duplex RNAs
one RNA strand associates with RISC-like protein
complex
21 base RNAs in active complexes
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Step Four. RISC-like complex anneals with
complementary RNA, regulating or degrading the
RNA and/or silencing the corresponding chromatin.
cytoplasm
chromatinsilencing
nucleus
RISC-like
5
mature mRNA
5
AAAA 3
nascent RNA
RISC
DNA
nucleosomes
RNApolymerase
mRNA attenuation,degradation, or activation
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Six easy steps to understanding RNAi
Step Five. Match with mRNA determines
degradation or attenuation.
mRNA
mRNA
cap
cap
AAAAA
ORF
AAAAA
ORF
imperfect match
perfect match
attenuation
degradation
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Six easy steps to understanding RNAi
Step Six. Our genome encodes hundreds of
regulatory microRNAs.
mRNA
ORF
cap
AAAAA
unprocessed micro RNA (miRNA) transcript
1. miRNA excision by "dicer"-like enzymes
imperfect match
2. assembly into RISC-like complex
translational attenuation (usually)
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Six easy steps to understanding RNAi
Step Six (and a half). Cell cycle control of
miRNA function.
FXR1
cell cycle arrest
AGO2
mRNA
miR369-3
TNF?
(AUUUA)n
cap
AAAAA
unprocessed micro RNA (miRNA) transcript
1. miRNA excision by "dicer"-like enzymes
imperfect match
2. assembly into RISC-like complex
translational activation upon cell cycle arrest
translational repression during growth
Vasudevan, Tong, and J Steitz (2007) Science, in
press.
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Contemporary evolutionary advantages of microRNAs
(miRNAs)
Independent transcriptional regulation of the
four mRNAscan be preserved while all four mRNAs
are coregulatedpost-transcriptionally as a
eukaryotic "regulon"
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Step Four. RISC-like complex anneals with
complementary RNA, regulating or degrading the
RNA and/or silencing the corresponding chromatin.
cytoplasm
chromatinsilencing
nucleus
RISC-like
5
mature mRNA
5
AAAA 3
nascent RNA
RISC
DNA
nucleosomes
RNApolymerase
mRNA attenuation,degradation, or activation
31
Six easy steps to understanding RNAi
Step One. A perfect RNA duplex is usually a sign
of danger.
bidirectional transcription of a compact viral
genome
RNA
viral DNA genome
RNA
transcription of dispersed repeated retroelements
("genomic parasites")
Alu RNA
L1 RNA
gene
genomic DNA
Alu
L1
L1
Alu
Alu
Alu RNA
Alu RNA
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de novo silencing
maintenance of silencing
transcription
DNA replication, proteins dissociate
dsRNA formation
RNAi amplification by RNA-dependent RNA polymerase
RNAi-mediated processing
proteins rebind to methylated histones
(worms not humans)
recruitment of histone methyltransferase
histone H3 K9 methylation
methylation spreads, DNA resilenced
recruitment of chromodomain proteins
Dernburg and Karpen (2002) Cell 111, 159-162
Coulmenares et al. (2007) Mol Cell 27, 449-461
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About 145 distinct human miRNAs have been
identified to date, and appear to regulate 20
of human genes Bartel (2004) MicroRNAs
Genomics, Biogenesis, Mechanism, and Function.
Cell 116, 281 Xie et al. (2005) Nature 434, 338.
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(No Transcript)
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Genes and Development 20, 2202-2207 (2006)
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Nature 435, 828-833 (2005)
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(No Transcript)
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Varieties of retrotransposons