The Regulation of Neuronal Gene Expression through
Alternative Pre-mRNA Splicing
Our lab is interested in the regulation of pre-mRNA splicing and the biochemical
mechanisms that control changes in splice sites. The sequences of metazoan
genomes with their relatively low gene numbers have highlighted the question of
how protein number can be expanded beyond the gene number for a complex
organism. Alternative splicing, in allowing the production of multiple mRNAs
and hence multiple proteins from a single gene, is a major contributor to
protein diversity. However, in spite of its key role in gene expression, this
process is poorly understood mechanistically.
Alternative splicing is particularly
common in genes expressed in the mammalian nervous system, where many proteins
important for neuronal differentiation and function are made in diverse
isoforms through controlled changes in splicing. Our lab works on a range of
projects related to the control of pre-mRNA splicing in neurons. We aim to
determine the mechanisms of action of splicing regulators and to understand
their roles in neural development. We are focused on four regulatory factors:
Polypyrimidine Tract Binding Protein (PTB), neuronal PTB, Fox-1, and Fox-2. PTB
and nPTB are primarily splicing repressors, while Fox-1 and Fox-2 act to
enhance splicing. Each of these proteins alters the splicing of a specific set
of exons within the genome. In mechanistic studies, we examine the RNA binding
properties of these proteins and analyze how they can alter spliceosome
assembly. A second effort uses cell culture models and conditional knockout
mice to understand how these proteins affect neuronal development. A final area
of interest is in understanding how cell signaling pathways impact the splicing
reaction. This project focuses on the effect of cell excitation on the splicing
of ion channel transcripts and the role of this splicing in neuronal
Mechanisms of Splicing Repression and Derepression
Our early work used the neural-specific
N1 exon of the c-src gene as a model for a tissue specific
splicing event. Using a variety of approaches, we dissected the molecular
components that determine c-src N1 exon splicing and its repression in
nonneuronal cells. Analyses of the cis-acting RNA elements controlling the exon
demonstrated that the tissue specificity of N1 derives from a combination of
enhancer and repressor sequences in the surrounding RNA. In neurons, an
enhancer sequence in the downstream intron activates splicing of the exon.
Splicing is specifically repressed in non-neuronal cells by sequences in the
upstream 3′ splice site and within the enhancer region. This combination of
positive and negative regulation has proven to be a common feature in systems
of tissue-specific splicing.
an in vitro splicing system developed in the lab to study the molecules that
regulate N1 splicing. Splicing of N1 is repressed in nuclear extracts of
non-neuronal HeLa cells. In contrast, splicing proceeds in extracts of WERI-1
retinoblastoma cells. Using this system, we have made particular progress in
understanding the proteins that mediate repression of N1 exon splicing. This
repression requires CUCUCU repressor elements that are bound by the polypyrimidine
tract–binding protein (PTB). Depleting PTB from HeLa extracts using a specific
antibody, or from cells by RNA interference, shows that PTB is required to
prevent N1 exon splicing. PTB-binding sites both upstream and downstream from
N1 are needed for splicing repression, and the presence of the downstream sites
stabilizes PTB binding to the upstream sites. This implies that PTB assembles a
cooperative repressor complex that bridges the exon. We are currently analyzing
the structure of this complex and its interactions with the RNA. Fred Allain’s
lab (ETH, Zurich) has shown by NMR spectroscopy that all four domains of PTB
each bind a pyrimidine triplet. However, the orientation of two of these
domains indicates that at least one of these triplets cannot be contiguous with
the other three. This ability to form an RNA loop, which fits well with the
placement of the repressor elements surrounding N1 and other exons regulated by
PTB, is being incorporated into models of the PTB/RNA interaction needed for splicing
also taken a proteomic approach to understanding the mechanism of PTB mediated
splicing repression. We find that PTB blocks splicing at an early stage of
spliceosome assembly, prior to the formation of what is called the E complex.
The large pre-mRNP complexes that lead to the E complex were purified and their
components were identified by mass spectrometry. Under conditions of PTB
dependent splicing repression, we find that the essential splicing factor U2AF
is blocked from assembling into the spliceosome. Under the conditions of our
experiments, U2AF requires an interaction with components at the 5’ splice site
to assemble into the E complex. Thus, PTB may be specifically blocking this
interaction, a possibility we are currently investigating. We are very
interested in these and other results in the field, which indicate that PTB may
interact with a specific spliceosomal target in repressing splicing.
To repress splicing, CUCUCU
elements that bind PTB are needed both upstream and downstream of the N1 exon.
These elements are apparently sufficient to determine the differential splicing
between HeLa and WERI-1 extracts. However in other cells, additional sequences
are required for proper regulation of the exon. The downstream element UGCAUG
binds the Fox proteins (see below), and is essential for enhanced splicing in
neurons. The downstream region also contains additional regulatory elements
that assemble a multi-protein complex comprised of the hnRNP H and KSRP
proteins, as well as PTB. We are currently working to understand the roles of
these other factors and their interplay with the Fox and PTB proteins. The
structures of pre-mRNP complexes and the nature of their assembly are an
understudied problem. In addition to elucidating how these RNA/Protein
complexes affect splicing, we hope our biochemical analyses will shed light on
the general properties of these structures that affect many different RNA-dependent
Cellular Functions of Splicing Regulators
WERI-1 extract, where PTB activity is reduced, an additional protein is present
called nPTB. Neuronal PTB is 74% identical to PTB in sequence, but is
specifically expressed in neurons in the brain, whereas PTB is mostly excluded
from neurons and expressed in glia. Neuronal PTB binds to the same CUCUCU
regulatory elements as PTB, but is apparently neutral for N1 exon splicing. It
does not repress splicing of neuronal exons in vitro and only mildly affects
their splicing in vivo. In contrast, other exons seem to be equally repressed
by PTB and nPTB. In addition to the cellular activities nPTB and its effects on
splicing in vitro, we are studying how and when the choice is made during
neuronal differentiation to express PTB or nPTB. Most interestingly, we find
that nPTB expression is regulated post-transcriptionally. NPTB mRNA is present
in most if not all cells, but this mRNA produces protein only in specific cell
types, notably neurons and testis. In many cells, nPTB expression is repressed
by PTB protein, presenting an interesting cross-regulation of one protein by
the other. In other cells, specific microRNAs repress nPTB expression. We are
currently characterizing this translational regulation of nPTB in more detail.
To characterize the roles of PTB and nPTB in neuronal
cell biology, we are developing mouse strains carrying alleles of these genes
that can be conditionally knocked out in particular cell lineages. PTB and nPTB
apparently regulate overlapping but not identical sets of exons. To identify
these exon sets and examine their cellular roles, we and others are developing
microarrays of splice-junction-specific oligonucleotides to assay large numbers
of alternative exons in parallel. We are using these arrays in conjunction with
the gene knockouts in mice, and with RNA interference mediated PTB knockdown,
to identify large sets of PTB regulated exons. The ability to do genome wide
analyses of alternative splicing will help us in a number of projects.
The Fox proteins are cell-type specific splicing
The enhancer region downstream of N1 is a complex
array of RNA regulatory elements that are required both for splicing
enhancement and for splicing repression. The most significant element for the
enhancer activity is the hexanucleotide UGCAUG, which is also essential for
other splicing enhancers and is found downstream of a whole set of
neuronally-regulated exons. This key hexanucleotide element is recognized by
several mammalian homologs of the C. elegans Fox-1 protein. We found that these RNA
binding proteins are strong activators of splicing for certain neuronal exons.
Through the use of multiple promoters and alternative splicing events, the
mouse Fox-1 and Fox-2 genes each encode a large family of related proteins that
are expressed in muscle, heart, and neurons. Interestingly, the human homolog
of Fox-1 (called A2BP1 for Ataxin 2 binding protein) is implicated in
cerebellar ataxias, as well as other forms of neurological disease. These
disorders may arise from problems of splicing control. Thus, in addition to
their mechanisms of splicing enhancement, we are extremely interested in the
cellular roles of the Fox proteins and how Fox regulated exons might affect
neuronal development and function. Using similar approaches to nPTB, we are
studying the Fox proteins in cell culture and in conditional knockout mice.
The Regulation of Ion Channel Alternative Splicing by
poorly understood aspect of splicing regulation is how extracellular stimuli
and cell signaling pathways direct changes in splicing. This is especially
interesting in the nervous system, where many proteins that determine neuronal
excitation are modulated by alternative splicing. To study how specific
signaling cascades alter splicing patterns, we examined the regulation of the
STREX exon in the calcium-activated potassium channel transcript (BK channel).
BK channels are important in shaping the action potential kinetics of a number
of excitable cells and are highly regulated at the level of splicing. The STREX
exon adds a peptide insert into the calcium-binding domain of the channel that
both makes it more sensitive to intracellular calcium and voltage and allows
its modulation by Protein Kinase A. We found that culturing excitable cells in
depolarizing media containing high potassium led to the repression of STREX
exon inclusion. This reduction in STREX splicing is blocked by the CaM Kinase
inhibitor KN93 and by the L-type calcium channel blocker nifedipine,
implicating calcium signaling in this splicing repression. As a model for this
regulation, we expressed the STREX exon from a transfected minigene and found
that STREX splicing is repressed by a cotransfected constitutive CaMK IV. This
approach allowed us to map specific RNA regulatory elements in the BK channel
transcript needed to respond to the CaMK signal. We identified a
calcium-responsive RNA element (CaRRE-1) that can confer CaMK-dependent
repression on a heterologous exon. We are very interested in the mechanism of
this repression and how it responds to culture conditions.
CaMK effect on ion channel splicing has implications for the control of
neuronal excitability. Alternative splicing alters the activity of many other
proteins that mediate the induction and propagation of action potentials. If
splicing is controlled by cell excitation, then these splicing events are
likely one component of the plastic changes in cell activity that underlie many
aspects of neurophysiology. Additional CaRRE elements were also found in two
regulated exons of the NMDA Receptor 1, which are also regulated by CaMK IV.
NR1 exon 21 contains a CaRRE element within the exon itself, instead of the 3’
splice site, and encodes a peptide that controls the trafficking and assembly
of the NR1 subunit. Exon 21 also contains an additional Calcium responsive
element, different from the CaRRE-1, which we are currently characterizing.
These elements in ion channel exons provide a unique entry point for the
dissection of signaling pathways that impinge on the splicing apparatus, and we
are working to identify the proteins that bind to them. In an alternative
approach, we are studying whether known splicing regulators can be altered in
expression or activity by specific signaling pathways. For example, we have
found that the nucleo/cytoplasmic distribution of PTB is regulated by
phosphorylation by Protein Kinase A. Our goal in these projects is to understand
how the information of an extracellular stimulus can be transmitted to the cell
nucleus to alter splicing.
For more information, please visit the Black Lab Website
- Pandya-Jones A., Bhatt D.M., Lin C.H., Tong A.J., Smale S.T., Black D.L. “Splicing kinetics and transcript release from the chromatin compartment limit the rate of Lipid A-induced gene expression.” RNA. 2013 Apr 24.
- Bhatt D.M., Pandya-Jones A., Tong A.J., Barozzi I., Lissner M.M., Natoli G., Black D.L., Smale S.T. “Transcript dynamics of proinflammatory genes revealed by sequence analysis of subcellular RNA fractions.” Cell. 2012 Jul 20;150(2):279-90.
- Keppetipola N., Sharma S., Li Q., Black D.L. “Neuronal regulation of pre-mRNA splicing by polypyrimidine tract binding proteins. PTBP1 and PTBP2.” Crit Rev Biochem Mol Biol. 2012 Jul-Aug;47(4):360-78.
- Anderson E.S., Lin C.H. Xiao X., Stoilov P., Burge C.B., Black D.L. “The cardiotonic steroid digitoxin regulates alternative splicing through depletion of the splicing factors SRSF3 and TRA2B.” RNA. 2012 May;18(5):1041-9. Epub 2012 Mar 28.
- Gehman L.T., Meera P., Stoilov P., Shiue L., O’Brien J.E., Meisler M.H., Ares M. Jr., Otis T.S., Black D.L. “The splicing regulator Rbfox2 is required for both cerebellar development and mature motor function.” Genes Dev. 2012 Mar 1;26(5):445-60.
- Zheng S., Gray E.E., Chawla G., Porse B.T., O’Dell T.J., Black D.L. “PSD-95 is post-transcriptionally repressed during any early neural development by PTBP1 and PTBP2.” Nat Neurosci. 2012 Jan 15;15(3):381-8.
- Gehman L.T., Stoilov P., Maguire J., Damianov A., Lin C.H., Shiue L., Ares M. Jr., Mody I., Black D.L., “The splicing regulator Rbfox1 (A2BP1) controls neuronal excitation in the mammalian brain,” Nat Genet. 2011 May 29;43(7):706-11.
- Sharma S., Maris C., Allain F.H., Black D.L., “U1 snRNA directly interacts with polypyrimidine tract-binding protein during splicing repression,” Mol Cell. 2011 Mar 4;41(5):579-88.
- Tang Z.Z., Sharma S., Zheng S., Chawla G., Nikolic J., Black D.L., “Regulation of the mutually exclusive exons 8a and 8 in the CaV1.2 calcium channel transcript by polypyrimidine tract-binding protein,” J Biol Chem. 2011 Mar 25; 286(12):10007-16. Epub 2011 Jan 31
- Black D.L., Gorospe M. “Tapas and RNA in Renaissance Spain.” RNA Biol. 2012 Mar-Apr;7(2);130-2.