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Lim KH, Ferraris L, Filloux ME, Raphael BJ, Fairbrother WG. Using Positional Distribution to Identify Splicing Elements and Predict pre-mRNA Processing Defects in Human Genes. PNAS 2011 [Epub Jun 17th]
Ferraris L, Stewart AP, Kang J, Desimone AM, Gemberling M, Tantin D, Fairbrother WG. Combinatorial binding of Transcription Factors in the Pluripotency Control Regions of the Genome. Genome Research 2011 April 28th
Ferraris L, Stewart AP, Gemberling MP, Reid DC, Lapadula MJ, Thompson WA, Fairbrother WG. Hig-throughput Mapping of Protein Occupancy Identifies Functional Elements without the Restriction of a Candidate Factor Approach. Nucleic Acids Research 2011 Mar;39(6):e33
Chang B, Levin J, Thompson WA, Fairbrother WG. High-Throughput Binding
Analysis Determines the Binding Specificity of ASF/SF2 on Alternatively Spliced
Human pre-mRNAs. Comb Chem High Throughput Screen. 2009 Dec 16. [Epub ahead of
print] PubMed PMID: 20015017.
Watkins KH, Stewart A, Fairbrother W. A rapid high-throughput method for
mapping ribonucleoproteins (RNPs) on human pre-mRNA. J Vis Exp. 2009 Dec 2;(34).
pii: 1622. doi: 10.3791/1622. PubMed PMID: 19956082.
Reid DC, Chang BL, Gunderson SI, Alpert L, Thompson WA, Fairbrother WG.
Next-generation SELEX identifies sequence and structural determinants of splicing
factor binding in human pre-mRNA sequence. RNA. 2009 Dec;15(12):2385-97. Epub
2009 Oct 27. PubMed PMID: 19861426; PubMed Central PMCID: PMC2779669.
Kang J, Gemberling M, Nakamura M, Whitby FG, Handa H, Fairbrother WG, Tantin
D. A general mechanism for transcription regulation by Oct1 and Oct4 in response
to genotoxic and oxidative stress. Genes Dev. 2009 Jan 15;23(2):208-22. PubMed
PMID: 19171782; PubMed Central PMCID: PMC2648538.
Tantin D, Gemberling M, Callister C, Fairbrother WG. High-throughput
biochemical analysis of in vivo location data reveals novel distinct classes of
POU5F1(Oct4)/DNA complexes. Genome Res. 2008 Apr;18(4):631-9. Epub 2008 Jan 22.
Erratum in: Genome Res. 2009 Apr;19(4):690. Fairbrother, William [corrected to
Fairbrother, William G]. PubMed PMID: 18212089; PubMed Central PMCID: PMC2279250.
Fairbrother WG, Lipscombe D. Repressing the neuron within. Bioessays. 2008
Jan;30(1):1-4. Review. Erratum in: Bioessays. 2009 Apr;31(4):487. Fairbrother,
Will [corrected to Fairbrother, William G]. PubMed PMID: 18081004; PubMed Central
nucleotide polymorphism-based validation of exonic
Center for Cancer Research, Massachusetts
Institute of Technology, Cambridge, Massachusetts, USA.
Because deleterious alleles arising from mutation are
filtered by natural selection, mutations that create
such alleles will be underrepresented in the set of
common genetic variation existing in a population at any
given time. Here, we describe an approach based on this
idea called VERIFY (variant elimination reinforces
functionality), which can be used to assess the extent
of natural selection acting on an oligonucleotide motif
or set of motifs predicted to have biological activity.
As an application of this approach, we analyzed a set of
238 hexanucleotides previously predicted to have exonic
splicing enhancer (ESE) activity in human exons using
the relative enhancer and silencer classification by
unanimous enrichment (RESCUE)-ESE method. Aligning the
single nucleotide polymorphisms (SNPs) from the public
human SNP database to the chimpanzee genome allowed
inference of the direction of the mutations that created
present-day SNPs. Analyzing the set of SNPs that overlap
RESCUE-ESE hexamers, we conclude that nearly one-fifth
of the mutations that disrupt predicted ESEs have been
eliminated by natural selection (odds ratio = 0.82 +/-
0.05). This selection is strongest for the predicted
ESEs that are located near splice sites. Our results
demonstrate a novel approach for quantifying the extent
of natural selection acting on candidate functional
motifs and also suggest certain features of mutations/SNPs,
such as proximity to the splice site and disruption or
alteration of predicted ESEs, that should be useful in
identifying variants that might cause a biological
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identifies candidate exonic splicing enhancers in
Center for Cancer Research, Massachusetts Institute of
Technology, Cambridge, MA 02139, USA.
A typical gene contains two levels of information: a
sequence that encodes a particular protein and a host of
other signals that are necessary for the correct
expression of the transcript. While much attention has
been focused on the effects of sequence variation on the
amino acid sequence, variations that disrupt gene
processing signals can dramatically impact gene
function. A variation that disrupts an exonic splicing
enhancer (ESE), for example, could cause exon skipping
which would result in the exclusion of an entire exon
from the mRNA transcript. RESCUE-ESE, a computational
approach used in conjunction with experimental
validation, previously identified 238 candidate ESE
hexamers in human genes. The RESCUE-ESE method has
recently been implemented in three additional species:
mouse, zebrafish and pufferfish. Here we describe an
online ESE analysis tool (http://genes.mit.edu/burgelab/rescue-ese/)
that annotates RESCUE-ESE hexamers in vertebrate exons
and can be used to predict splicing phenotypes by
identifying sequence changes that disrupt or alter
identification of exonic splicing enhancers in human
Department of Biology, Center for Cancer
Research, Massachusetts Institute of Technology,
Cambridge, MA 02139, USA.
Specific short oligonucleotide sequences that enhance
pre-mRNA splicing when present in exons, termed exonic
splicing enhancers (ESEs), play important roles in
constitutive and alternative splicing. A computational
method, RESCUE-ESE, was developed that predicts which
sequences have ESE activity by statistical analysis of
exon-intron and splice site composition. When large data
sets of human gene sequences were used, this method
identified 10 predicted ESE motifs. Representatives of
all 10 motifs were found to display enhancer activity in
vivo, whereas point mutants of these sequences exhibited
sharply reduced activity. The motifs identified enable
prediction of the splicing phenotypes of exonic
mutations in human genes.
genomic sequences that inhibit splicing.
Department of Biological Sciences,
Columbia University, New York, New York 10027, USA.
Mammalian genes are characterized by relatively small
exons surrounded by variable lengths of intronic
sequence. Sequences similar to the splice signals that
define the 5' and 3' boundaries of these exons are also
present in abundance throughout the surrounding introns.
What causes the real sites to be distinguished from the
multitude of pseudosites in pre-mRNA is unclear. Much
progress has been made in defining additional sequence
elements that enhance the use of particular sites. Less
work has been done on sequences that repress the use of
particular splice sites. To find additional examples of
sequences that inhibit splicing, we searched human
genomic DNA libraries for sequences that would inhibit
the inclusion of a constitutively spliced exon. Genetic
selection experiments suggested that such sequences were
common, and we subsequently tested randomly chosen
restriction fragments of about 100 bp. When inserted
into the central exon of a three-exon minigene, about
one in three inhibited inclusion, revealing a high
frequency of inhibitory elements in human DNA. In
contrast, only 1 in 27 Escherichia coli DNA fragments
was inhibitory. Several previously identified silencing
elements derived from alternatively spliced exons
functioned weakly in this constitutively spliced exon.
In contrast, a high-affinity site for U2AF65 strongly
inhibited exon inclusion. Together, our results suggest
that splicing occurs in a background of repression and,
since many of our inhibitors contain splice like
signals, we suggest that repression of some pseudosites
may occur through an inhibitory arrangement of these