Sika Zheng

PSD-95 is post-transcriptionally repressed during early neural development by PTBP1 and PTBP2.

Postsynaptic density protein 95 (PSD-95) is essential for synaptic maturation and plasticity. Although its synaptic regulation has been widely studied, the control of PSD-95 cellular expression is not understood. We found that Psd-95 was controlled post-transcriptionally during neural development. Psd-95 was transcribed early in mouse embryonic brain, but most of its product transcripts were degraded. The polypyrimidine tract binding proteins PTBP1 and PTBP2 repressed Psd-95 (also known as Dlg4) exon 18 splicing, leading to premature translation termination and nonsense-mediated mRNA decay. The loss of first PTBP1 and then of PTBP2 during embryonic development allowed splicing of exon 18 and expression of PSD-95 late in neuronal maturation. Re-expression of PTBP1 or PTBP2 in differentiated neurons inhibited PSD-95 expression and impaired the development of glutamatergic synapses. Thus, expression of PSD-95 during early neural development is controlled at the RNA level by two PTB proteins whose sequential downregulation is necessary for synapse maturation.

Alternative pre-mRNA splicing in neurons: growing up and extending its reach

Alternative pre-mRNA splicing determines the protein output of most neuronally expressed genes. Many examples have been described of protein function being modulated by coding changes in different mRNA isoforms. Several recent studies demonstrate that, through the coupling of splicing to other processes of mRNA metabolism, alternative splicing can also act as an on/off switch for gene expression. Other regulated splicing events may determine how an mRNA is utilized in its later cytoplasmic life by changing its localization or translation. These studies make clear that the multiple steps of post-transcriptional gene regulation are strongly linked. Together, these regulatory process play key roles in all aspects of the cell biology of neurons, from their initial differentiation, to their choice of connections, and finally to their function with mature circuits.

The splicing regulator PTBP2 controls a program of embryonic splicing required for neuronal maturation

We show that the splicing regulator PTBP2 controls a genetic program essential for neuronal maturation. Depletion of PTBP2 in developing mouse cortex leads to degeneration of these tissues over the first three postnatal weeks, a time when the normal cortex expands and develops mature circuits. Cultured Ptbp2−/− neurons exhibit the same initial viability as wild type, with proper neurite outgrowth and marker expression. However, these mutant cells subsequently fail to mature and die after a week in culture. Transcriptome-wide analyses identify many exons that share a pattern of mis-regulation in the mutant brains, where isoforms normally found in adults are precociously expressed in the developing embryo. These transcripts encode proteins affecting neurite growth, pre- and post-synaptic assembly, and synaptic transmission. Our results define a new genetic regulatory program, where PTBP2 acts to temporarily repress expression of adult protein isoforms until the final maturation of the neuron. DOI: http://dx.doi.org/10.7554/eLife.01201.001

The neurogenetics of alternative splicing

Alternative precursor-mRNA splicing is a key mechanism for regulating gene expression in mammals and is controlled by specialized RNA-binding proteins. The misregulation of splicing is implicated in multiple neurological disorders. We describe recent mouse genetic studies of alternative splicing that reveal its critical role in both neuronal development and the function of mature neurons. We discuss the challenges in understanding the extensive genetic programmes controlled by proteins that regulate splicing, both during development and in the adult brain.

A hierarchical Bayesian model for comparing transcriptomes at the individual transcript isoform level

The complexity of mammalian transcriptomes is compounded by alternative splicing which allows one gene to produce multiple transcript isoforms. However, transcriptome comparison has been limited to differential analysis at the gene level instead of the individual transcript isoform level. High-throughput sequencing technologies and high-resolution tiling arrays provide an unprecedented opportunity to compare transcriptomes at the level of individual splice variants. However, sequence read coverage or probe intensity at each position may represent a family of splice variants instead of one single isoform. Here we propose a hierarchical Bayesian model, BASIS (Bayesian Analysis of Splicing IsoformS), to infer the differential expression level of each transcript isoform in response to two conditions. A latent variable was introduced to perform direct statistical selection of differentially expressed isoforms. Model parameters were inferred based on an ergodic Markov chain generated by our Gibbs sampler. BASIS has the ability to borrow information across different probes (or positions) from the same genes and different genes. BASIS can handle the heteroskedasticity of probe intensity or sequence read coverage. We applied BASIS to a human tiling-array data set and a mouse RNA-seq data set. Some of the predictions were validated by quantitative real-time RT–PCR experiments.

Developmental Control of CaV1.2 L-Type Calcium Channel Splicing by Fox Proteins

ABSTRACT CaV1.2 voltage-gated calcium channels play critical roles in the control of membrane excitability, gene expression, and muscle contraction. These channels show diverse functional properties generated by alternative splicing at multiple sites within the CaV1.2 pre-mRNA. The molecular mechanisms controlling this splicing are not understood. We find that two exons in the CaV1.2 channel are controlled in part by members of the Fox family of splicing regulators. Exons 9* and 33 confer distinct electrophysiological properties on the channel and show opposite patterns of regulation during cortical development, with exon 9* progressively decreasing its inclusion in the CaV1.2 mRNA over time and exon 33 progressively increasing. Both exons contain Fox protein binding elements within their adjacent introns, and Fox protein expression is induced in cortical neurons in parallel with the changes in CaV1.2 splicing. We show that knocking down expression of Fox proteins in tissue culture cells has opposite effects on exons 9* and 33. The loss of Fox protein increases exon 9* splicing and decreases exon 33, as predicted by the positions of the Fox binding elements and by the pattern of splicing in development. Conversely, overexpression of Fox1 and Fox2 proteins represses exon 9* and enhances exon 33 splicing in the endogenous CaV1.2 mRNA. These effects of Fox proteins on exons 9* and 33 can be recapitulated in transfected minigene reporters. Both the repressive and the enhancing effects of Fox proteins are dependent on the Fox binding elements within and adjacent to the target exons, indicating that the Fox proteins are directly regulating both exons. These results demonstrate that the Fox protein family is playing a key role in tuning the properties of CaV1.2 calcium channels during neuronal development.

Regulation of the mutually exclusive exons 8a and 8 in the CaV1.2 calcium channel transcript by polypyrimidine tract binding protein

CaV1.2 calcium channels play roles in diverse cellular processes such as gene regulation, muscle contraction, and membrane excitation and are diversified in their activity through extensive alternative splicing of the CaV1.2 mRNA. The mutually exclusive exons 8a and 8 encode alternate forms of transmembrane segment 6 (IS6) in channel domain 1. The human genetic disorder Timothy syndrome is caused by mutations in either of these two CaV1.2 exons, resulting in disrupted Ca2+ homeostasis and severe pleiotropic disease phenotypes. The tissue-specific pattern of exon 8/8a splicing leads to differences in symptoms between patients with exon 8 or 8a mutations. Elucidating the mechanisms controlling the exon 8/8a splicing choice will be important in understanding the spectrum of defects associated with the disease. We found that the polypyrimidine tract-binding protein (PTB) mediates a switch from exon 8 to 8a splicing. PTB and its neuronal homolog, nPTB, are widely studied splicing regulators controlling large sets of alternative exons. During neuronal development, PTB expression is down-regulated with a concurrent increase in nPTB expression. Exon 8a is largely repressed in embryonic mouse brain but is progressively induced during neuronal differentiation as PTB is depleted. This splicing repression is mediated by the direct binding of PTB to sequence elements upstream of exon 8a. The nPTB protein is a weaker repressor of exon 8a, resulting in a shift in exon choice when nPTB replaces PTB in cells. These results provide mechanistic understanding of how these two exons, important for human disease, are controlled.

NMDA-induced neuronal survival is mediated through nuclear factor I-A in mice

Identification of the signaling pathways that mediate neuronal survival signaling could lead to new therapeutic targets for neurologic disorders and stroke. Sublethal doses of NMDA can induce robust endogenous protective mechanisms in neurons. Through differential analysis of primary library expression and microarray analyses, here we have shown that nuclear factor I, subtype A (NFI-A), a member of the NFI/CAAT-box transcription factor family, is induced in mouse neurons by NMDA receptor activation in a NOS- and ERK-dependent manner. Knockdown of NFI-A induction using siRNA substantially reduced the neuroprotective effects of sublethal doses of NMDA. Further analysis indicated that NFI-A transcriptional activity was required for the neuroprotective effects of NMDA receptor activation. Additional evidence of the neuroprotective effects of NFI-A was provided by the observations that Nfia(-/-) neurons were highly sensitive to NMDA-induced excitotoxicity and were more susceptible to developmental cell death than wild-type neurons and that Nfia(+/-) mice were more sensitive to NMDA-induced intrastriatal lesions than were wild-type animals. These results identify NFI-A as what we believe to be a novel neuroprotective transcription factor with implications in neuroprotection and neuronal plasticity following NMDA receptor activation.

Identify Alternative Splicing Events Based on Position-Specific Evolutionary Conservation

The evolution of eukaryotes is accompanied by the increased complexity of alternative splicing which greatly expands genome information. One of the greatest challenges in the post-genome era is a complete revelation of human transcriptome with consideration of alternative splicing. Here, we introduce a comparative genomics approach to systemically identify alternative splicing events based on the differential evolutionary conservation between exons and introns and the high-quality annotation of the ENCODE regions. Specifically, we focus on exons that are included in some transcripts but are completely spliced out for others and we call them conditional exons. First, we characterize distinguishing features among conditional exons, constitutive exons and introns. One of the most important features is the position-specific conservation score. There are dramatic differences in conservation scores between conditional exons and constitutive exons. More importantly, the differences are position-specific. For flanking intronic regions, the differences between conditional exons and constitutive exons are also position-specific. Using the Random Forests algorithm, we can classify conditional exons with high specificities (97% for the identification of conditional exons from intron regions and 95% for the classification of known exons) and fair sensitivities (64% and 32% respectively). We applied the method to the human genome and identified 39,640 introns that actually contain conditional exons and classified 8,813 conditional exons from the current RefSeq exon list. Among those, 31,673 introns containing conditional exons and 5,294 conditional exons classified from known exons cannot be inferred from RefSeq, UCSC or Ensembl annotations. Some of these de novo predictions were experimentally verified.

A broadly applicable high-throughput screening strategy identifies new regulators of Dlg4 (Psd-95) alternative splicing

Most mammalian genes produce multiple mRNA isoforms derived from alternative pre-mRNA splicing, with each alternative exon controlled by a complex network of regulatory factors. The identification of these regulators can be laborious and is usually carried out one factor at a time. We have developed a broadly applicable high-throughput screening method that simultaneously identifies multiple positive and negative regulators of a particular exon. Two minigene reporters were constructed: One produces green fluorescent protein (GFP) from the mRNA including an exon, and red fluorescent protein (RFP) from the mRNA lacking the exon; the other switches these fluorescent products of exon inclusion and exclusion. Combining results from these two reporters eliminates many false positives and greatly enriches for true splicing regulators. After extensive optimization of this method, we performed a gain-of-function screen of 15,779 cDNA clones and identified 40 genes affecting exon 18 of Discs large homolog 4 (Dlg4; also known as post-synaptic density protein 95 [Psd-95]). We confirmed that 28 of the 34 recoverable clones alter reporter splicing in RT-PCR assays. Remarkably, 18 of the identified genes encode splicing factors or RNA binding proteins, including PTBP1, a previously identified regulator of this exon. Loss-of-function experiments examining endogenous Dlg4 transcripts validated the effects of five of eight genes tested in independent cell lines, and two genes were further confirmed to regulate Dlg4 splicing in primary neurons. These results identify multiple new regulators of Dlg4 splicing, and validate an approach to isolating splicing regulators for almost any cassette exon from libraries of cDNAs, shRNAs, or small molecules.