Multiple control Mechanisms & Involvement in human disease
Anabella Srebrow
Laboratorio de Fisiología y Biología Molecular IFIBYNE-CONICET Facultad de Ciencias Exactas y Naturales Universidad de Buenos Aires outside
cytoplasm
nucleus protein-coding genes
Cleavage and Polyadenylation Capping Splicing Co-transcriptional processing
post-transcriptional co-transcriptional pre-mRNA processing pre-mRNA processing
Pol II Pol II Transcription Capping Transcription Splicing Polyadenylation Primary transcript
CAP Capping Splicing Polyadenylation
An CAP An CAP Co-transcriptional processing
• requires coordination between molecular machineries acting at different steps
• coordination is mainly achieved by the function of RNA polymerase II
CTD (Carboxi Terminal Domain) multiple repeats of “YSPTSPS” heptad Serine phosphorylations are critical for transcription & association with processing factors mRNA factory governed by pol II CTD
capping polyadenylation factors factors P P P P
P P P
Splicing factors CTD
(YSPTSPS)52
Bentley lab: McCracken et al., Nature 1997 Figure 6-23 Molecular Biology of the Cell (© Garland Science 2008) Pol II CTD is crucial for pre-mRNA processing
TRANSCRIPTION CAPPING SPLICING polyA
Pol III Normal Inhibited Inhibited Inhibited
Cleveland lab: Sisodia et al., MCB 1987
Normal Normal Normal Normal
Normal* Inhibited Inhibited Inhibited
Bentley lab: McCracken et al., Nature 1997 What is Splicing ?
AUG STOP
Lynch, Nature Reviews Immunol. 2004 Sequences required for splicing
30-50 nt exon exon a c c a c ...AGgu agu...... //.... x u a ...//…………..ccucuucucuucuccuxcagG..... g u u g u Branch site Polypyrimidine ’ tract 5 splice site ’ (5’ss) 3 splice site (3’ss) DONOR SITE ACCEPTOR SITE The Spliceosome
snRNP snRNP snRNP U5 U4 U6
snRNP U1 snRNP U2AF65 U2AF35 U2 BBP 30-50 nt exon exon a c c a c ...AGgu agu...... //.... x u a ...//…………..ccucuucucuucuccuxcagG..... g u u g u Branch site Polypyrimidine ’ tract 5 splice site ’ (5’ss) 3 splice site (3’ss) DONOR SITE ACCEPTOR SITE Snurps or snRNPs
• Small nuclear ribonucleoprotein particles (small nuclear RNA + proteins)
• U1, U2, U4, U5 and U6 involved in nuclear RNA splicing
• Snurps constitute components of the spliceosome and are key components of splicing process
• Each snurp has a unique role is splicing and the sequence of the RNA component of the snurp determines the role played in splicing alowing specific snRNA-premRNA interactions Downloaded from cshperspectives.cshlp.org on October 5, 2011 - Published by Cold Spring Harbor Laboratory Press
C.L. Will and R. Lu¨hrmann
E A 1 p
O A OH H + 5¢SS BP 3¢SS p E1 p A ppE2 A E2 E1 p E2 Step 1 Step 2
B Branch Poly Y 5¢ splice site 3¢ splice site site tract The spliceosome cycle Exon GURAGU YNCURAC Y(n) YAG Exon Metazoans
Exon GUAUGU UACUAAC YAG Exon Yeast
C
5¢ SS BP 3¢ SS GU A AG Pre-mRNA Intron mRNA U1 Prp43 - ATP Brr2 - ATP Snu114 - GTP U1 U2
Complex E U5 U6 U2 U2 Prp5 - ATP U5 U6 Sub2/UAP56 - ATP Prp22 - ATP
U1 U2 U6 U2 Pre-spliceosome U6 U4 (complex A) U5 U5 Post-spliceosomal Prp28 - ATP U4/U6.U5 complex tri-snRNP U1 Prp22 - ATP Prp16 - ATP U6 U4 2nd step
U1 U5 U2 U6 Pre-catalytic U4 U2 spliceosome U5 Catalytic step 1 (complex B) Brr2 - ATP spliceosome (complex C) Snu114 - GTP U6 U2 U6 U2 1st step
U5 U5 Bact B* (activated) Prp2 - ATP (catalytically activated)
D
SR protein
U2AF RS U2 U1 65 35 RRM
Exon GU A YYYYYY AG ESE Exon GU Wahl, Will & Luhrmann, 2011 Luhrmann, & Will Wahl,
Figure 1. Pre-mRNA splicing by theU2-typespliceosome. (A) Schematicrepresentation of thetwo-step mechanism of pre-mRNA splicing. Boxes and solid linesrepresent theexons(E1, E2) and theintron, respectively. Thebranch site adenosineisindicated by theletter A and thephosphategroups( p) at the5′ and 3′ splicesites, which ar econser ved in the splicing products, ar e also shown. (Seefacing pagefor legend.)
4 Cite as Cold Spring Harb Perspect Biol 2011;3:a003707 Dynamic composition of spliceosome
Wahl, Will & Luhrmann, 2011 Other proteins (SR proteins) cooperate with snRNPs for spliceosome assembly Three types of spliceosome
In mammalian cells Trypanosomes and nematodes More than 99% Less than 1%
Figure 6-34a Molecular Biology of the Cell (© Garland Science 2008) What is Alternative Splicing ?
splice site sequences may deviate from the consensus recognition sites for the spliceosome, creating “weak splice sites” (less efficiently recognized by spliceosome)
COMPETITION between different donor or different acceptor sites
Recognition of splice sites by the spliceosome can be impaired by pre-mRNA secondary structure or by RNA binding proteins (splicing regulatory factors)
Differential removal of exonic sequences or inclusion of intronic ones
Different alternative splicing patterns
mRNA pre-mRNA isoforms
constitutive
exon cassette
A mutually exclusive exons A B B Alternative alternative 5’splice sites Splicing
alternative 3’ splice sites
A intron retention The relevance of alternative splicing
It is more a rule than an exception. It is estimated to affect the expression of more than 65% of human genes. It explains how a vast protein diversity is achieved with a limited number of genes. Mutations that affect alternative splicing regulatory sequences are a widespread source of human disease.
Examples of alternative splicing
AUG Thyroid AATAA AATAA A A 2 3 3 4 5 6
AUG Neurons fruitless (sex-specific AS) Calcitonin/CGRP transcription factor - droshophila sex hormone determination
epithelial tissue Embryo VASE 7 8 7 IIIb IIIc 10 Adult mesenchymal tissue NCAM (develpmentally regultated FGFR-2 AS) GF receptor (diff. binding properties) neural cell adhesion molecule Smith and Valcárcel, TIBS 2000 Splicing regulatory proteins
Nilsen & Graveley, Nature 2010, vol 463 Key regulators of alternative splicing
They bind to regulatory sequences (ENHANCERS or SILENCERS) within exons or introns, stimulating or inhibiting inclusion of alternative regions into mRNA
SR PROTEINS (serine/arg rich)
hnRNP PROTEINS (Heterogenous nuclear ribonucleoproteins)
Lynch, Nature Reviews Immunol. 2004 The balance of positive- and negative-acting factors present in a given cell determine the extent of regulated exon inclusion
ISS intronic splicing silencer; ISE intronic splicing enhancer ESS exonic splicing silencer; ESE exonic splicing enhancer Regulation of splicing factor activity by extracellular signals
extracellular stimuli
signalling pathways (ERK, Akt, JNK)
Expression level Post-translational modification Subcelular-localization of splicing factors
Regulation of pre-mRNA alternative splicing Mutations that affect alternative splicing regulatory sequences are a widespread source of human disease. 15-50% of the mutations that cause genetic disease affect the splicing process -thalassemia abnormal hemoglobin abnormal processsing of -globin due to point mutations
Figure 6-35 Molecular Biology of the Cell (© Garland Science 2008) Examples of mutations in cis-acting sequences that are associated with malignant disease
Gene Disease Mutated sequence Molecular Reference consequences LKB1 Peutz-Jeghers Disruption of minor (tumour suppressor) Syndrome IVS2+1A>G spliceosome Hasting et al., 2005 (increased cancer risk) 5’ splice site. Lower protein levels
KIT Gastrointestinal Deletion of 9 Aberrant splicing Chen et al., 2005 (oncogene) stromal tumour nucleotides including Constitutive protein IVS10 3’ splice site activation
LI-Cadherin Hepatocellular IVS6+35 A>G Generation of a ISS? Wang et al., 2005 carcinoma Exon 7 skipping
LI-Cadherin Hepatocellular E6 codon 651 Generation of a ESS? Wang et al., 2005 carcinoma Exon 7 skipping
KLF6 Prostate cancer IVSDA Generation of binding Narla et al., 2005 (tumour suppressor) site (ISE) for SRp40 Novel splicing variants act as dominant negatives
HAS1 Multiple myeloma E3 C7760T Exon 4 skipping Adamia et al. 2005 (hyaluronan synthase)
BRCA1 Breast and ovarian E18 G5199T [E1694X] ESE disruption Mazoyer et al. 1998 (tumour suppressor) cancer Exon 18 skipping
S Table 1. Examples of mutations in splicing cis-acting sequences that are associatedSrebrow with malignant & Kornblihtt disease. 2006, JCS Mutations disrupting cis-acting splicing sequences cause splicing defects and disease
SMN protein: important for snRNP biogenesis SMN deficiencies = Motor neuron degenerative disease
Mutation creates a splicing silencer, leading to exon skipping and a partially functional dystrophin protein. Dystrophin communicates the extracellular matrix with the contractile apparatus in skeletal muscle cells
Tau: microtubule associated protein important for neuronal processes (neurite outgrowth/axonal transport) ex 10 = 4th microtubule binding domain altered ratio, aggregation of Tau neurodegenerative disease
Transmembrane Chloride Channel important for proper function of secretory
epithelium (lung, intestine, testes) Cooper, Wan and and Dreyfuss.Wan Cell 2009, 136: 777 Cooper, Minor splicing, Disrupted
700-800 genes in the human genome contain U12-type introns
Pessa & Frilander, 2011 Science Mutations in the snRNA component of the “minor U4” (U4atac) linked to human disease (developmental disorder MOPD I or TALS) interaction between human U6atac and U4atac snRNAs, mutations identified in U4atac
mutations distort the conserved stem-loop structure, preventing the binding of spliceosomal protein Genetic alterations of the major splicing components linked to human pathogenesis Mutations in multiple components of the splicing machinery associated to myelodysplasia, would lead to impaired recognition of 3’ss and the production of aberrantly spliced mRNAs
Yoshida et al., Nature 2011 Epithelial-Mesenchymal Transition involves changes in alternative splicing (Rac1, Ron, CD44, FGFR...) and can be triggered by splicing changes
EMT epithelial cells mesenchymal cells
EMT •loss of cell adhesions •loss of epithelial phenotype •adquisition of mesenchymal phenotype •adquisition of migratory and invasive capacity
MET
Thiery and Sleeman, Nature Reviews 2006 The SR protein SRSF1 promotes skipping of exon 11 of the Ron proto-oncogene to produce constitutively active isoform that confers invasive phenotype
+ Ron (normal RTK)... 10 11 12 epithelial phenotype 10 12
SRSF1
Ron pre-mRNA 10 11 12
SRSF1 SRSF1SRSF1 SRSF1 SRSF1
10 11 12 + ∆Ron (constitutively active RTK) ... 10 12 mesenchymal phenotype, invasive growth
Ghigna et al., Mol Cell 2005 - Valacca et al., JCB 2010
Spinal Muscular Atrophy (SMA) caused by low levels of SMN protein (required for snRNP assembly) & a possible therapy
fu functional
SMN protein
delivering antisense oligonucleotides to inhibit the binding of
fu SMN1 gene SMN1 duplication of of duplication a splicing repressor to an intronic splicing silencer (in intron 7) induces exon 7 inclusion and production of higher levels of SMN functional protein from SMN2 gene