Programs of alternative splicing regulation by polypyrimidine tract binding protein

Programs of alternative splicing regulation by polypyrimidine tract binding protein

Funding Type: 
Basic Biology II
Grant Number: 
RB2-01502
Award Value: 
$1,344,562
Stem Cell Use: 
Embryonic Stem Cell
Status: 
Active
Public Abstract: 
The therapeutic promise of stem cell biology lies in its potential for cell replacement therapies in diseases where an essential cell type of the patient malfunctions or degenerates. This is particularly evident in diseases of the nervous system where cells largely lose their ability to proliferate and thus regenerate after embryonic differentiation. Devastating neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), are characterized by a progressive paralysis caused by motor neuron death and currently have no cure. Strategies for replacing specific neuronal cell types with cells derived from human embryonic stem (hES) cells will require understanding the genetic programs that control hES cell differentiation. These rapidly dividing pluripotent cells undergo a major transition in gene expression to become neuronal progenitor cells (NPC), while maintaining their proliferative ability. Another drastic change in gene expression program occurs as NP cells differentiate into neurons, where cell division has stopped. A great deal of important work is describing the DNA level changes that control gene expression in ES cells and during their transition to NPC and neurons. However, the production of a protein product from a gene is controlled at each step in the gene expression pathway where the DNA gene is first transcribed into RNA and the RNA then translated into protein. An important RNA level regulatory step in this pathway is the processing of the primary RNA transcript from the gene into an mRNA that can be translated into protein. One part of this processing is the pre-mRNA splicing reaction, where alternative splicing patterns in the pre-mRNA determine the structure of the final protein product of most human genes. Little is known about how this step in the gene expression pathway is regulated in ES cells or during their differentiation. Yet ALS and SMA can both be caused by the loss of components of the splicing machinery and a great deal of work is examining how splicing might be disrupted in mature neurons of ALS and SMA patients. In this study, we will examine how two important splicing regulators, the polypyrimidine tract binding protein (PTB) and its neuronal homolog nPTB, affect splicing in normal ES and NP cells. We will characterize the programs of regulation controlled by these proteins. In particular, we will focus on those parts of the PTB regulated splicing program that affect cell proliferation and the ability of ES and NP cells to self renew. From this work, we will advance our understanding of how ES cells differentiate into neurons and how pre-mRNA splicing controls cell function in normal development and in disease.
Statement of Benefit to California: 
Neurological diseases affect millions of patients in California and elsewhere. For example, spinal muscular atrophy (SMA) is one of the most common genetic causes of infant death and has no effective treatment. SMA and other neurological diseases are caused by errors in the cellular process of pre-mRNA splicing. One promising strategy for neurological treatments is in cell replacement therapies using hES and iPS cells as source material for regenerating normal neurons in place of those lost to the disease. Another therapeutic strategy for SMA is in drugs that alter the splicing process to improve its efficiency in diseased cells. This project will examine the splicing process in normal hES cells and how it is regulated when these cells differentiate into neuronal progenitor cells and neurons. This will provide essential information on the biology of stem cells needed to move towards various therapeutic applications. The project will also provide a system for drug discovery in the new field of splicing targeted therapeutics. This work will help California to continue to lead in these areas of basic research, as well as provide the state with a head start in biotechnology and pharmaceutical development for the practical application of these discoveries.
Progress Report: 

Year 1

The therapeutic promise of stem cell biology lies in its potential for cell replacement therapies in diseases where an essential cell type of the patient malfunctions or degenerates. This is particularly evident in diseases of the nervous system where cells largely lose their ability to proliferate and thus regenerate after embryonic differentiation. Devastating neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), are characterized by a progressive paralysis caused by motor neuron death and currently have no cure. Strategies for replacing specific neuronal cell types with cells derived from human embryonic stem (hES) cells will require understanding the genetic programs that control hES cell differentiation. These rapidly dividing pluripotent cells undergo a major transition in gene expression to become neuronal progenitor cells (NPC), while maintaining their proliferative ability. Another drastic change in gene expression program occurs as NP cells differentiate into neurons, where cell division has stopped. A great deal of work has described the DNA level changes that control gene expression in ES cells and during their transition to NPC and neurons. However, the production of a protein product from a gene is controlled at each step in the gene expression pathway where the DNA gene is first transcribed into RNA and the RNA then translated into protein. An important RNA level regulatory step in this pathway is the processing of the primary RNA transcript from the gene into an mRNA that can be translated into protein. One part of this processing is the pre-mRNA splicing reaction, where alternative splicing patterns in the pre-mRNA determine the structure of the final protein product of most human genes. Little is known about how this step in the gene expression pathway is regulated in ES cells or during their differentiation. Yet ALS and SMA can both be caused by the loss of components of the splicing machinery and a great deal of work is examining how splicing might be disrupted in mature neurons of ALS and SMA patients. In this study, we are examining how two important splicing regulators, the polypyrimidine tract binding protein (PTB) and its neuronal homolog nPTB, affect splicing in normal ES and NP cells. We are characterizing the programs of regulation controlled by these proteins. In the past funding period we adapted and applied two new genomewide methods of RNA analysis. The first uses new technologies for high density RNA sequencing (RNAseq) to examine the whole transcriptome of each cell under study. From this data, we can extract information on all the splicing changes occurring during a developmental transition. The second method called CLIP examines the sites of RNA binding by PTB and nPTB in the RNA of each cell type. These methods are now ready to apply to ESC, NPC and motor neurons that we have derived in culture. From this work, we will advance our understanding of how ES cells differentiate into neurons and how pre-mRNA splicing controls cell function in normal development and in disease.

Year 2

The therapeutic promise of stem cell biology lies in its potential for cell replacement therapies in diseases where an essential cell type of the patient malfunctions or degenerates. This is particularly evident in diseases of the nervous system where cells largely lose their ability to proliferate and thus regenerate after embryonic differentiation. Devastating neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), are characterized by a progressive paralysis caused by motor neuron death and currently have no cure. Strategies for replacing specific neuronal cell types with cells derived from human embryonic stem (hES) cells will require understanding the genetic programs that control hES cell differentiation. These rapidly dividing pluripotent cells undergo a major transition in gene expression to become neuronal progenitor cells (NPC), while maintaining their proliferative ability. Another drastic change in gene expression program occurs as NP cells differentiate into neurons, where cell division has stopped. A great deal of work has described the DNA level changes that control gene expression in ES cells and during their transition to NPC and neurons. However, the production of a protein product from a gene is controlled at each step in the gene expression pathway where the DNA gene is first transcribed into RNA and the RNA then translated into protein. An important RNA level regulatory step in this pathway is the processing of the primary RNA transcript from the gene into an mRNA that can be translated into protein. One part of this processing is the pre-mRNA splicing reaction, where alternative splicing patterns in the pre-mRNA determine the structure of the final protein product of most human genes. Little is known about how this step in the gene expression pathway is regulated in ES cells or during their differentiation. Yet ALS and SMA can both be caused by the loss of components of the splicing machinery and a great deal of work is examining how splicing might be disrupted in mature neurons of ALS and SMA patients. In this study, we are examining how two important splicing regulators, the polypyrimidine tract binding protein (PTB) and its neuronal homolog nPTB, affect splicing in normal ES and NP cells. We are characterizing the programs of regulation controlled by these proteins. In the past two funding periods, we adapted and applied two new genomewide methods of RNA analysis. The first uses new technologies for high density RNA sequencing (RNAseq) to examine the whole transcriptome of each cell under study. From this data, we have extracted information on all the splicing changes occurring during a developmental transition. The second method called CLIP identifies the sites of RNA binding by PTB and nPTB in the RNA of each cell type. These methods have been applied to hESC, NPC and motor neurons that we have derived in culture. From this work, we learning new events determining how ES cells differentiate into neurons and how pre-mRNA splicing controls cell function in normal development and in disease.

Year 3

The therapeutic promise of stem cell biology lies in its potential for cell replacement therapies in diseases where a particular cell type or cellular function has been lost. For example, the devastating neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) are characterized by a progressive loss of motor neurons and consequent paralysis. Cell replacement for diseases of the nervous system poses special challenges because many neurons lose their ability to proliferate and thus regenerate after embryonic differentiation. Strategies for replacing specific neuronal cell types with cells derived from human embryonic stem (hES) cells will require understanding the genetic programs that control hES cell differentiation. These rapidly dividing pluripotent cells undergo a major transition in gene expression to become neuronal progenitor cells (NPC), while maintaining their proliferative ability. A second dramatic change in the program of gene expression occurs when these progenitor cells cease cell division and differentiate into neurons. A great deal of work has described the DNA level changes affecting gene expression in ES cells and during their transition to NPC and neurons. However, the gene expression pathway is also highly regulated at the RNA level, as the DNA gene is transcribed into RNA and the RNA then translated into protein. An important regulatory step in this pathway is the processing of the primary RNA transcript from the gene into an mRNA that can be translated into protein. This processing includes pre-mRNA splicing, and alternative splicing patterns in the pre-mRNA determine the final protein output of most human genes. Little is known about how this step in the gene expression pathway is regulated in ES cells or during their differentiation. Yet ALS and SMA can both be caused by the loss of components of the splicing machinery and a great deal of work is examining how splicing might be disrupted in mature neurons of ALS and SMA patients. In this study, we have examined how two important splicing regulators, the polypyrimidine tract binding protein (PTBP1) and its neuronal homolog PTBP2, affect splicing in normal ES and NP cells, and have characterized their regulatory networks. We adapted and applied two new technologies for RNA analysis. The first used high density RNA sequencing (RNAseq) to examine the whole transcriptome of each cell under study. From this data, we extracted information on all the splicing changes occurring during a developmental transition. The second method called CLIP identifies the sites of binding by PTBP1 and PTBP2 in the RNA of each cell type. These methods were applied to hESC, NPC and motor neurons that we have derived in culture. From this work, we identified many new genetic events that determine how ES cells differentiate into neurons. These alternative splicing events controlled by the PTB proteins affect numerous functions essential to the development and survival of neurons. We are now examining several of new genetic regulatory events in more detail to understand how pre-mRNA splicing controls cell function in normal development and in disease.

© 2013 California Institute for Regenerative Medicine