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Multitasking Rett Protein Shines Spotlight on RNA Splicing in Neurologic and Psychiatric Disease

28 October 2005. A protein that underlies a severe and fairly common neurodevelopmental disease called Rett syndrome has a more sophisticated function than scientists previously suspected. Called MeCP2, the protein was thought to silence gene transcription in a process involving DNA methylation and chromatin remodeling. This process, in turn, serves as an example mechanism for the broader issue of epigenetic control of gene expression. As if this was not interesting enough, however, researchers led by Huda Zoghbi at Baylor College of Medicine in Houston report in the 26 October PNAS Early Edition that MeCP2 also regulates the RNA splicing of certain genes. The two roles of MeCP2 might even be coupled, the authors speculate. When MeCP2 comes off the promoter of a target gene to permit its expression, perhaps it promptly turns around to the pre-mRNA that can now peel off the DNA and helps control which exons go into the final mRNA, thereby influencing the sequence of the translated protein, the scientists suggest.

This study is not directly relevant to schizophrenia, but all the same, it is worth reading because it exemplifies a new research direction as scientists try to make deeper inroads into the complexities of the molecular pathophysiology of neurologic and psychiatric diseases. Mutations in the gene for MeCP2 have come up in the study of other neurodevelopmental diseases including autism. More broadly, MeCP2 is a neural protein known to become abundant during childhood when synapses mature, suggesting that it is important for activity-dependent plasticity.

At the basic level, interest in how proteins interact with RNA to orchestrate the splicing of pre-mRNA is being fanned by the humbling realization coming out of the Human Genome Project that humans contain fewer than 30,000 genes, probably no more than simpler creatures such as the puffer fish. The human proteome is much larger than its genome, however, implying that an expansion of variety must occur at the level of post-transcriptional processes. Part of that variety arises from regulated changes in splicing, which generate different RNA isoforms from the same pre-mRNA. By orchestrating which exons go into the final protein, alternative splicing can multiply the number of proteins that can be manufactured from a given gene. Many labs now study the proteins that oversee the cutting and pasting necessary for that. Some focus on how the splicing and transcription machineries are coupled, others focus on how alternative splicing controls formation of the synapse (Ule et al., 2005), and yet others explore how alternative splicing gives rise to tissue-specific isoforms of a given protein. One cutting edge in this area that is relevant to SRF readers concerns the question of how neuronal activity, signaling, and other aspects of the molecular context of a neuron are linked to RNA splicing (e.g., Beffert et al., 2005).

The theme of RNA processing cuts across emerging pathophysiologies of a number of neurologic and psychiatric diseases. Examples include Alzheimer disease, spinomuscular atrophy, myotonic dystrophy, schizophrenia, and fragile X mental retardation. While RNA splicing abnormalities are not root causes of these diseases, splicing shows up in various guises in all of them. In AD research, for example, it has long been known that certain splice variants of tau are important in the development of tau pathology (D’Souza and Schellenberg, 2005). Earlier this year, researchers reported that a new candidate gene for late-onset AD might exert its influence on risk through alternative splicing (Bertram et al., 2005). Others suggested that ineffective splice variants of the human IDE gene (Farris et al., 2005) and more active splice variants of BACE might play a role (Zohar et al., 2005).

RNAs for most channels and receptor proteins are alternatively spliced. That includes metabotropic glutamate receptors, proteins that are implicated in neurologic and psychiatric diseases including mental retardation and schizophrenia (Weinberger, 2005; Niswender et al., 2005). With regard to schizophrenia, alternative splicing has been suggested for two of the susceptibility genes that draw increasing support in the field, i.e., neuregulin and ZDHHC8 (Kirov et al., 2005).

In the present study, Zoghbi's team focused on the protein underlying Rett syndrome, which affects one in every 10,000 girls and a smaller percentage of boys. While the phenotype varies, babies generally develop normally for a year or so and then regress, growing up with mental retardation, abnormal movements including a characteristic hand wringing, seizures, and without being able to speak or socialize. A break in the understanding of this baffling disease came when Zoghbi’s lab identified causal mutations in the MeCP2 gene located on the X chromosome (Amir et al., 1999). Such mutations show up on the autism spectrum, too, but favorable X chromosome inactivation patterns can lead to a milder phenotype in these cases.

Researchers immediately tried to find out exactly how this protein functions. Early studies showed that MeCP2, which stands for methyl-CpG-binding protein 2, not only binds to methylated cytosines in DNA, but also associates with a repressor complex containing histone deacetylases. This pointed toward a global role in silencing transcription, but things soon became complicated. When scientists compared transcriptional profiles of MeCP2 knockout mice with those from wild-type, they saw no clear-cut gene expression changes even though the knockout mice did have the Rett phenotype (Tudor et al., 2002). Moreover, researchers for years had difficulty identifying MeCP2 target genes. When several labs recently did manage to find some, the picture only became murkier because each seemed to be regulated by MeCP2 in a different way, not by a common mechanism of transcriptional repression. For example, BDNF turned out to be an activity-dependent target of MeCP2 that was repressed in the classic way of MeCP2 occupying its promoter (Chen et al., 2003; Martinovich et al., 2003). By contrast, repression of the target gene Dlx5 had to do with genetic imprinting and required formation of a silent chromatin loop (Horike et al., 2005). This and other data led Zoghbi to believe that no single known mechanism for MeCP2 could explain Rett pathogenesis to date and that a new, unbiased functional analysis was needed.

Toward this goal, first author Juan Young and colleagues searched for proteins that interact with MeCP2. Immunoprecipitation and mass spectrometry studies identified Y box binding protein 1 (YB-1), an evolutionarily conserved DNA and RNA binding protein that had been previously implicated in alternative splicing, regulation or transcription and translation, DNA repair, and other cellular functions. RNA was necessary to maintain the interaction between MeCP2 and YB-1. Prior work on YB-1 led them to suspect that the MeCP2-YB-1 complex serves to coordinate splicing with gene transcription by pulling YB-1 to nascent transcripts after MeCP2 is released from a gene promoter, the authors write.

The scientists first confirmed this idea by measuring the splicing of a reporter minigene in transfected cells. Next, the scientists picked a candidate neuronal gene and tested whether its splicing depends on the Rett protein. They chose the NMDA receptor subunit NR1 because of the previously reported link between neuronal activity and MeCP2 targets. An alternative splice site in exon 22 of this gene’s mRNA is known to generate different protein variants in response to activity (Mu et al., 2003). Comparing brain tissue from Rett knockout and from wild-type mice, the scientists found differences in the distribution of the splice variants in subcortical areas but not in cerebral cortex, hinting that the NR1 pre-mRNA might be a target for MeCP2 only in certain brain areas.

Finally, the researchers took a more global look at splicing changes. Using a custom-made microarray carrying probes specific to individual exons and exon-exon junctions, they performed a genomewide survey of splicing changes in cerebral cortex mRNA from a Rett MeCP2 mouse model and wild-type mice. They report changes in alternative splicing of 54 genes, most of them classic cassette exon changes. When clustered, the changes classified the genotype of the samples. (This data is published as supplemental material on the PNAS website.) Validation of the candidate transcripts identified with the array showed that 35 percent of them were spliced abnormally in cerebral cortex of other MeCP2 mutant mice. One of them was Dlx5, a gene previously identified as a MeCP2 target and known to have at least seven splice forms.

In summary, the study paints a more intricate portrait of MeCP2 as a protein with multiple functions in neurons. It raises the question whether Rett syndrome is as much a disorder of RNA splicing as of DNA expression. Given that different proteins can roll off a defective splicing machinery, studying errors in this process might lead scientists toward a better understanding of why doctors see such heterogeneous symptoms in patients who share a given genetic defect, in this case, mutations in the gene MeCP2.—Gabrielle Strobel.

Young JI, Hong EP, Castle JC, Crespo-Barreto J, Bowman AB, Rose MF, Kang D, Richman R, Johnson JM, Berget S, Zoghbi HY. Regulation of RNA splicing by the methylation-dependent transcriptional repressor methyl-CpG binding protein 2. Proc Natl Acad Sci U S A. 2005 Oct 26; [Epub ahead of print]. Abstract

Comments on News and Primary Papers
Comment by:  Mary Reid
Submitted 6 February 2006
Posted 8 February 2006

Gabrielle Strobel mentions that DLX5 has been suggested as an MECP2 target. Horike and colleagues report that "transcription of DLX5 and DLX 6 was reproducibly approximately two times higher in Mecp2-null mice" (Holrike et al., 2005). In view of the fact that DLX5 is necessary for osteoblastogenesis and osteoclastogenesis, might we suspect that its increased expression may explain the bone pathology reported in Rett syndrome? Does this resemble Paget disease and if so, might we expect that biphosphonates may be beneficial in Rett syndrome?

Horike S, Cai S, Miyano M, Cheng JF, Kohwi-Shigematsu T. Loss of silent-chromatin looping and impaired imprinting of DLX5 in Rett syndrome. Nat Genet. 2005 Jan;37(1):31-40. Epub 2004 Dec 19. Abstract

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Related Paper: Sequence Variants in SLITRK1 Are Associated with Tourette's Syndrome.

Comment by:  Tom Insel
Submitted 17 October 2005
Posted 17 October 2005
  I recommend this paper

This is an important paper for at least two reasons. First, it demonstrates yet again the power of finding a rare, real genetic lesion. The inversion on 13q31.1 in a single case was the smoking gun in this story that led to the search for SLITRK1 variations in a pedigree. The second reason this paper should be studied by scientists searching for genes for mental illness is that the pathology involves a non-coding mutation. This is the first report of a mutation that affects a site for microRNA binding. First studied in C. elegans and more recently in mammals, microRNAs are endogenous 22 mer transcripts that either cause translational arrest or cleavage of mRNA transcripts. They target a region in the 3'UTR, in this case in the region just down from the coding sequence of SLITRK1. A frameshift mutation in this region would alter regulation by microRNA-189. Now the search can be launched to find the developmental link between microRNA-189 effects and the neural circuitry for the Tourette-ADHD phenotype.

Bartel DP and Chen CZ, Micromanagers of gene expression. Nat Rev Genetics 5:396, 2004. Abstract

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Related News: Gene Expression Study May Open Window on Brain Development

Comment by:  Barbara Lipska
Submitted 15 June 2009
Posted 15 June 2009

In this very important and innovative study, Sestan and colleagues report a transcriptome-wide survey across multiple brain regions of the fetal mid-gestation brain. They show dramatic differences in expressed transcripts, including alternative splice variants, between brain regions, and most surprisingly, between several cortical regions. The authors have undertaken an ambitious task of further characterizing differentially expressed genes by functional clustering and co-expression clustering and comparing the results with genes identified through neurobiological experiments. They have also performed extensive validation using several additional fetal brains. Most interestingly, the authors showed that differentially expressed genes are more frequently associated with human-specific evolution of putative cis-regulatory elements. For this, they have identified genes that are near highly conserved non-coding sequences (CNSs) and found that the genes that are differentially expressed between the regions are more frequently near human-specific accelerated evolution CNSs.

The weakness of the study is a very small number of fetal brains (four) and the fact that they range in age from 18 to 23 weeks of gestation. During these several weeks of fetal life, the brain undergoes dramatic developmental changes and expression of many genes, either increases or decreases steeply. Thus, it would be critical to fully characterize these changes across fetal age. It is also crucial to explore genetic influences on fetal gene expression as it appears that in adult brain both gene expression and splicing are strongly genetically regulated. The authors have made an important contribution to our understanding of development of human brain, and further research of this type will generate the data that would help in better understanding of human brain disorders. In particular, genetic-expression effects in human brain across the entire lifespan, including fetal period, may help identify molecular mechanisms whereby candidate genes increase risk for developing the disorder. Using expression levels of transcripts and their splicing characteristics as intermediate phenotypes may yield statistically positive associations and improved understanding of the mechanisms that lead to neurodevelopmental disorders such as autism and schizophrenia, as they are the most proximal phenotypes to the risk alleles.

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Related News: Gene Expression Study May Open Window on Brain Development

Comment by:  Karoly Mirnics, SRF Advisor
Submitted 15 June 2009
Posted 15 June 2009

This outstanding study reinforces how much we still do not understand about human brain development and function! It is just mind-boggling that the mid-fetal human brain expresses more than three quarters of the human genome, and that region-specific splicing appears to be an absolutely critical feature of the developing brain. Interestingly, the structural and functional interhemispheric differences do not appear to be related to gene expression differences in mid-fetal life, but rather, either they develop independently of gene expression patterns, or they are developing at later stages of cortical maturation, perhaps in a postnatal activity-driven pattern.

So, how is this developmental expression machinery related to various neurodevelopmental disorders, such as schizophrenia? Is usage of an "inappropriate" splice variant sufficient to alter the neuronal phenotypic development to a degree that would predispose the brain to developing a disease? Are environmental insults capable of disrupting this finely tuned, region-specific splicing machinery? As this is a likely possibility, we must rethink the existing disease-related gene expression findings in the context of the present study, and accept that our previous gene expression measurements may have been too crude to uncover some of the most meaningful changes that are potentially hallmarks of various brain disorders. Furthermore, as the genes that show the most widespread regional use of splice variants can be essential for proper neuronal migration or connectivity, one can argue that these genes should be the primary targets for evaluation in the various regions of postmortem tissue of diseased individuals.

Finally, there is also a minor, cautionary note arising from this study. The fact that the Affymetrix U133 and the Exon array results showed a correlation of R2 >0.5 is encouraging, but underscores that platform-dependence of the findings remains a significant interpretational challenge. Some platforms will be better suited to identify certain gene expression changes, while others will have a greater power to reveal a different (but also potentially valid!) set of mRNA alterations.

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