Rinn & Chang Ann. Rev. Biochem 81:145, 2012

“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 cell-type specific) Nature 489:101, 2012

RNA content in eukaryotic cells

Total cellular RNA

~ 4% mRNA

and small regulatory RNAs (eg. snRNA, snoRNA, miRNA, siRNA…)

Adapted from Imam Nucl. Acids Res. 38:1559, 2009

Probe: gene X

Nucl Cyto

~ 96% non-coding RNAs (mostly rRNA and tRNA)

Northern blot analysis using RNA isolated from cytoplasm vs. nucleus

“Traditional” view of gene expression in eukaryotes

Zhang Nature Reviews Genetics 3: 698, 2002

Why is this picture out-of-date?

Montes et al. Gene 501:104, 2012

CTD code and pre-mRNA processing

CTD - 52 heptad repeats (of YSPTSPS) in humans, 26 in yeast

dynamic changes in its phosphorylation profile “Cracking the CTD code”

PIC: pre-initiation complex

Types of splicing

1. Nuclear pre-mRNA (spliceosomal)

2. Nuclear pre-tRNA

3. Group I intron

4. Group II intron

5. Archaeal pre-tRNA, pre-rRNA

2-step transesterification& lariat excised intron

endonuclease & ligase machinery

- self-splicing

- self-splicing

- two-step transesterification & lariat intron

(autocatalytic, ribozyme)

Cis-elements in spliceosomal introns

Wahl et al Cell 136: 701, 2009

(autocatalytic, ribozyme)

- mobile genetic element

- mobile genetic retroelement

Splicing of pre-mRNAs via two transesterification reactions

OH

Brown Fig.10.14

2’OH

Same biochemical pathway in group II intron ribozymes

1st step

2nd step

McManus & Gravely Curr.Opin.Gen.Dev 21:373, 2011

U1 snRNA base pairs with 5’ splice site

U2AF-65kD protein binds pyrimidine tract

U2 snRNA base pairs with branchpoint site

U2AF-35kD protein recognizes AG at 3’ splice site

SR proteins (“Ser-Arg” rich splicing activators) bind enhancers (ESE or ISE) & recruit other splicing factors…

BBP (or SF1 in mammals) – branchpoint bridging protein

hnRNPs (repressors) bind silencers (ESS or ISS)Irimia & Blencowe Curr Opin Cell Biol 24:323, 2012

How to determine that specific nucleotides at splice site are important?

Time course of splicing for labelled pre-mRNAs in HeLa cell nuclear extract

Gaur Proc Natl Acad Sci 97:115 (2000)

Site-directed mutation (at A of 3’splice site) and monitor effect using in vitro splicing assay

Montes et al. Gene 501:104, 2012

Step-wise assembly of spliceosomal machinery

3D structure by cryo-EM Azubel Mol Cell Sept 2004

How many proteins does the spliceosome contain?

Conformational rearrangements during spliceosomal assembly

Wahl et al Cell 136: 701, 2009

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

RRM: RNA recognition motif

RS: Arg-Ser rich motif

“Gel mobility shift” or “gel retardation” assays

One way to monitor specific RNA-protein interactions:

Native, non-denaturing gelelectrophoresis & autoradiography

1 2

Incubate protein extract 5’ 3’

* * *

* * *

Unlabelled RNA competitor

3 4

with labelled RNA

1 = RNA, no incubation (control)2 = RNA incubated with protein3 = 2 + “cold” competitor RNA4 = 2 + more competitor RNA

Cho PNAS 108:8233, 2011

Shen & Green Mol Cell 16:363, 2004

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

- TEV linker cleaved with specific protease

- RNase to degrade any RNA not protected by protein

- pre-mRNA labelled with single radioactive phosphate at 5’ ss or BP or...

How to detect protein-protein interactions?

- fusion protein (eg. GST-CTD) attached to resin- determine which proteins bind specifically to column

1. Affinity chromatography

Lodish Fig.3-43

Alberts Fig. 8-50

2. Co-immunoprecipitation

FRET Fluorescence resonance energy transfer (in vivo)

2 differentfluorophores (or quencher)

Chudakov Trends Biotech 12:605, 2005

RNA-RNA interactions

Protein-protein interactions

Will & Luhrmann Cold Spring Harbor Perspectives Biol 3:7, 2011

“only stem-loop 1 of U5 snRNA is shown”

- U1 and U4 snRNPs leave spliceosome

- U6 snRNA base-pairs with 5’ end of intron & restructuring of base-pairing between U6 and U2 snRNAs

Dynamic network of RNA interactions (snRNA-snRNA, snRNA-intron...) in spliceosome

Precatalytic spliceosome Catalytically activated spliceosome

Site-directed mutagenesis – loss of function

One way of examining specific, short RNA-RNA interactions:

followed by suppressor mutations – restoration of function

Smith Mol Cell 26:883, 2007

Assay to monitor effect on splicing(eg. in vitro splicing assay…)

U6 5’ …UGAUC…3’ | | | | |U2 3 ’…ACUAG…5’

wt Mutant

Double mutant

Is the spliceosome a ribozyme (like group II introns)?

.... or is splicing catalyzed by protein?

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

Candidate protein: Prp8 (in U5 snRNP)

Trans-splicing of pre-mRNAs

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)

- separate SL “spliced leader” RNA provides the first (non-coding) exon

- biochemical mechanism is fundamentally the same as cis-splicing

- excised intron is Y-shaped

- found in certain protists (eg. trypanosomes) and “lower” animals (eg. nematodes)

Blumenthal WormBook. 2005 pp1-9.

Landfear PNAS 100:7, 2003

In trypanosomes & nematodes, some genes are organized in operons !

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 (50SS) and branch point (A), respectively. The U2-auxiliary factor heterodimer (U2AF2/U2AF1) interacts with the polypyrimidine track (Y) and 30 splice site (30 SS), formingcomplex 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 ofthe catalytic complex (complex C). Two transesterification reactions join the exons and release an intron lariat that is subsequently degraded and the spliceosomecomponents 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 arereplaced by homologous U11, U12, U4atac, and U6atac snRNPs, respectively. The red star indicates the components that are mutated in neoplasias. The black star indicatesthe components that are mutated in retinitis pigmentosa. The orange star indicates the mutation in U4atac that is associated with microcephalic osteodysplastic primordialdwarfism type 1 (MOPD1). Abbreviations: ESEs, exonic splicing enhancers; ESSs, exonic splicing silencers

Singh & Cooper Trends Mol Med 18:472, 2012 2d Minor Class of Spliceosomal Introns

“AT-AC introns: an “ATtACk on dogma”

Mount, Science 271:1690, 1996