Transcript AT-AC introns: an

“The New RNA World”
“RNA molecules perform a stunningly diverse and elegantly complex set of functions”
2012
ENCODE project:
although only
~1.5% human
genome is
protein-coding,
up to 75% is
transcribed
(much being celltype specific)
Nature 489:101,
2012
Rinn & Chang Ann. Rev. Biochem 81:145, 2012
RNA content in eukaryotic cells
Total cellular RNA
~ 4% mRNA
~ 96% non-coding RNAs (mostly rRNA and tRNA)
and small regulatory RNAs (eg. snRNA,
snoRNA, miRNA, siRNA…)
Northern blot analysis using RNA isolated
from cytoplasm vs. nucleus
Nucl
Cyto
Probe: gene X
Adapted from Imam Nucl. Acids Res. 38:1559, 2009
“Traditional” view of gene expression in eukaryotes
Why is this picture out-of-date?
Zhang Nature Reviews Genetics 3: 698, 2002
CTD code and pre-mRNA processing
PIC: pre-initiation
complex
CTD - 52 heptad repeats (of YSPTSPS) in humans, 26 in yeast
dynamic changes in its phosphorylation profile
“Cracking the CTD code”
Montes et al. Gene 501:104, 2012
Types of splicing
1. Nuclear pre-mRNA (spliceosomal)
2-step transesterification & lariat excised intron
2. Nuclear pre-tRNA
endonuclease & ligase machinery
3. Group I intron
(autocatalytic, ribozyme)
- self-splicing
- mobile genetic element
4. Group II intron
- self-splicing (autocatalytic, ribozyme)
- mobile genetic retroelement
- two-step transesterification & lariat intron
5. Archaeal pre-tRNA, pre-rRNA
Cis-elements in spliceosomal introns
Wahl et al Cell 136: 701, 2009
Splicing of pre-mRNAs via two transesterification reactions
2’OH
1st step
OH
2nd step
Same biochemical pathway in group II intron ribozymes
Brown Fig.10.14
U1 snRNA base pairs with 5’ splice site
U2 snRNA base pairs with branchpoint site
U2AF-65kD protein binds pyrimidine tract
U2AF-35kD protein recognizes AG at 3’ splice site
BBP (or SF1 in mammals) – branchpoint bridging protein
SR proteins (“Ser-Arg” rich splicing activators) bind enhancers (ESE or ISE)
& recruit other splicing factors…
hnRNPs (repressors) bind silencers (ESS or ISS)
Irimia & Blencowe Curr Opin Cell Biol 24:323, 2012
McManus & Gravely Curr.Opin.Gen.Dev 21:373, 2011
How to determine that specific nucleotides at splice site are important?
Site-directed mutation (at A of 3’splice site)
and monitor effect using in vitro splicing assay
Time course of splicing for labelled pre-mRNAs
in HeLa cell nuclear extract
Gaur Proc Natl Acad Sci 97:115 (2000)
Step-wise assembly of spliceosomal machinery
3D structure by cryo-EM
Azubel Mol Cell Sept 2004
How many proteins does
the spliceosome contain?
Montes et al. Gene 501:104, 2012
Conformational rearrangements during spliceosomal assembly
RRM: RNA recognition motif
RS: Arg-Ser rich motif
U1 snRNP
U1 snRNP
- base-pairing between branchpoint region and U2 snRNA is stabilized by Arg-Ser
rich domain of U2AF65 and components of U2 snRNP
Wahl et al Cell 136: 701, 2009
One way to monitor specific RNA-protein interactions:
“Gel mobility shift” or “gel retardation” assays
Incubate protein extract
5’
with labelled RNA
1
2
3
***
3’
4
Native, non-denaturing gel
electrophoresis & autoradiography
1 = RNA, no incubation (control)
2 = RNA incubated with protein
3 = 2 + “cold” competitor RNA
4 = 2 + more competitor RNA
***
Cho PNAS 108:8233, 2011
Unlabelled RNA competitor
UV-crosslinking studies
- if RNA & protein physically very close, can
covalently link by UV treatment
- RS (Arg-Ser rich domain) &
RBD (RNA binding domain) of
U2AF65
- pre-mRNA labelled with single
radioactive phosphate at 5’ ss
or BP or...
- RNase to degrade any RNA not
protected by protein
- TEV linker cleaved with
specific protease
Shen & Green Mol Cell 16:363, 2004
How to detect protein-protein interactions?
1. Affinity chromatography
2. Co-immunoprecipitation
Lodish Fig.3-43
Alberts Fig. 8-50
- fusion protein (eg. GST-CTD) attached to resin
- determine which proteins bind specifically to column
FRET
Fluorescence resonance energy transfer (in vivo)
Protein-protein interactions
RNA-RNA interactions
Chudakov Trends Biotech 12:605, 2005
2 different
fluorophores
(or quencher)
Dynamic network of RNA interactions (snRNA-snRNA, snRNA-intron...)
in spliceosome
Precatalytic spliceosome
Catalytically activated spliceosome
- U1 and U4 snRNPs leave spliceosome
- U6 snRNA base-pairs with 5’ end of intron
& restructuring of base-pairing between U6
and U2 snRNAs
“only stem-loop 1 of U5 snRNA is shown”
Will & Luhrmann Cold Spring Harbor Perspectives Biol 3:7, 2011
One way of examining specific, short RNA-RNA interactions:
Site-directed mutagenesis – loss of function
followed by suppressor mutations – restoration of function
Assay to monitor effect on splicing
wt
U6 5’ …UGAUC…3’
|| | ||
U2 3 ’…ACUAG…5’
(eg. in vitro splicing assay…)
Mutant
Double mutant
Smith Mol Cell 26:883, 2007
Is the spliceosome a ribozyme (like group II introns)?
.... or is splicing catalyzed by protein?
Candidate protein: Prp8 (in U5 snRNP)
Prp8 can be crosslinked to 5’ SS, the branchpoint
region, and to 3’ SS
“Colored regions encompass
residues that exhibit interactions
with the factors indicated.”
Wahl et al Cell 136: 701, 2009
Trans-splicing of pre-mRNAs
- found in certain protists (eg. trypanosomes) and “lower” animals (eg. nematodes)
- separate SL “spliced leader” RNA provides the first (non-coding) exon
- biochemical mechanism is
fundamentally the same as cissplicing
- excised intron is Y-shaped
In C. elegans, ~ 70% mRNAs are trans-spliced and in trypanosomes virtually all
mRNAs are trans-spliced (so have the same 22 nt at end of 5’ UTRs for all mRNAs)
Blumenthal WormBook. 2005 pp1-9.
In trypanosomes & nematodes, some genes are organized in operons !
Landfear PNAS 100:7, 2003
2d Minor Class of Spliceosomal Introns
Singh & Cooper Trends Mol Med 18:472, 2012
“AT-AC introns: an
“ATtACk on dogma”
Mount, Science 271:1690, 1996
Figure 1. Spliceosome assembly and disease-associated mutations in spliceosome components. The broken lines and rectangles represent introns and exons, respectively.
The left panel shows the assembly of the major (U2 type) spliceosome. U1 and U2 small nuclear ribonucleoproteins (snRNPs) are recruited to the consensus 50 splice site (50
SS) and branch point (A), respectively. The U2-auxiliary factor heterodimer (U2AF2/U2AF1) interacts with the polypyrimidine track (Y) and 30 splice site (30 SS), forming
complex A. The U4/6 and U5 snRNPs join the assembling spliceosome followed by remodeling of the complex leading to removal of the U1 and U4 snRNP and formation of
the catalytic complex (complex C). Two transesterification reactions join the exons and release an intron lariat that is subsequently degraded and the spliceosome
components are recycled for subsequent rounds of splicing. The right panel shows the assembly of the minor (U12 type) spliceosome, in which U1, U2, U4, and U6 are
replaced by homologous U11, U12, U4atac, and U6atac snRNPs, respectively. The red star indicates the components that are mutated in neoplasias. The black star indicates
the components that are mutated in retinitis pigmentosa. The orange star indicates the mutation in U4atac that is associated with microcephalic osteodysplastic primordial
dwarfism type 1 (MOPD1). Abbreviations: ESEs, exonic splicing enhancers; ESSs, exonic splicing silencers