Role of the NMD RNA Decay Pathway in Maintaining the Stem-Like State

A key quality of stem cells is their ability to switch from a proliferative cell state in which they reproduce themselves to a differentiated cell state that ultimately allows them to carry out the functions of a fully mature cell. Most research on the nature of this switch has focused on the role of proteins that determine whether the genetic material—DNA—generates a copy of it itself in the form of messenger RNA, a process called transcription. In stem cells, such proteins—which are called transcription factors—activate the production of messenger RNAs encoding proteins that promote the proliferative and undifferentiated cell state. They also increase the production of messenger mRNAs that encode inhibitors of differentiation and cell proliferation. The level and profile of such transcription factors are altered in response to signals that trigger stem cells to differentiate. For example, transcription factors that promote the undifferentiated cell state are decreased in level and transcription factors that drive differentiation down a particular lineage are increased in level. While this transcription factor-centric view of stem cells explains some aspects of stem cell biology, it is, in of itself, insufficient to explain many of their behaviors, including both their ability to maintain the stem-like state and to differentiate. We hypothesize that a molecular pathway that complements transcription-base mechanisms in controlling stem cell maintenance vs. differentiation decisions is an RNA decay pathway called nonsense-mediated RNA decay (NMD). Messenger RNA decay is as important as transcription in determining the level of messenger RNA. Signals that trigger increased decay of a given messenger RNA leads to decreased levels of its encoded protein, while signals that trigger the opposite response increase the level of the encoded protein. Our project revolves around two main ideas. First, that NMD promotes the stem-like state by preferentially degrading messenger RNAs that encode differentiation-promoting proteins and proliferation inhibitor proteins. Second, that NMD must be downregulated in magnitude to allow stem cells to differentiate. During the progress period, we obtained substantial evidence for both of these hypotheses. With regard to the first hypothesis, we have used genome-wide approaches to identify hundreds of messenger RNAs that are regulated by NMD in both in vivo (in mice) and in vitro (in cell lines). To determine which of these messenger mRNAs are directly degraded by NMD, we have used a variety of approaches. This work has revealed that NMD preferentially degrades messenger RNAs encoding neural differentiation inhibitors and proliferation inhibitors in neural stem cells. In contrast, very few messenger RNAs encoding pro-stem cell proteins or pro-proliferation proteins are degraded by NMD. Together this provides support for our hypothesis that NMD promotes the stem-like state by shifting the proportion of messenger RNAs in a cell towards promoting an undifferentiated, proliferative cell state. With regard to the second hypothesis, we have found that many proteins that are directly involved in the NMD pathway are downregulated upon differentiation of stem and progenitor cells. Not only are NMD proteins reduced in level, but we find that the magnitude of NMD itself is reduced. We have used a variety of molecular techniques to determine whether this NMD downregulatory response has a role in neural differentiation and found that NMD downreglation is both necessary and sufficient for this event. Such experiments have also revealed particular messenger mRNAs degraded by NMD that are crucial for the NMD downregulatory response to promote neural differentiation. Our research has implications for intellectual disability cases in humans caused by mutations in an X-linked gene essential for NMD. Patients with mutations in this gene—UPF3B—not only have intellectual disability, but also schizophrenia, autism, and attention-deficit/hyperactivity disorder. Thus, the study of NMD may provide insight into a wide spectrum of cognitive and psychological disorders. We are currently in the process of generating induced-pluripotent stem (iPS) cells from intellectual disability patients with mutations in the UPF3B gene towards this goal.

A key quality of stem cells is their ability to switch from a proliferative cell state in which they reproduce themselves to a differentiated cell state that ultimately allows them to carry out the functions of a fully mature cell. Most research on the nature of this switch has focused on the role of proteins that determine whether the genetic material—DNA—generates a copy of it itself in the form of messenger RNA, a process called transcription. In stem cells, such proteins—which are called transcription factors—activate the production of messenger RNAs encoding proteins that promote the proliferative and undifferentiated cell state. They also increase the production of messenger mRNAs that encode inhibitors of differentiation and cell proliferation. The level and profile of such transcription factors are altered in response to signals that trigger stem cells to differentiate. For example, transcription factors that promote the undifferentiated cell state are decreased in level and transcription factors that drive differentiation down a particular lineage are increased in level. While this transcription factor-centric view of stem cells explains some aspects of stem cell biology, it is, in of itself, insufficient to explain many of their behaviors, including both their ability to maintain the stem-like state and to differentiate. We hypothesize that a molecular pathway that complements transcription-base mechanisms in controlling stem cell maintenance vs. differentiation decisions is an RNA decay pathway called nonsense-mediated RNA decay (NMD). Messenger RNA decay is as important as transcription in determining the level of messenger RNA. Signals that trigger increased decay of a given messenger RNA leads to decreased levels of its encoded protein, while signals that trigger the opposite response increase the level of the encoded protein. Our project revolves around two main ideas. First, that NMD promotes the stem-like state by preferentially degrading messenger RNAs that encode differentiation-promoting proteins and proliferation inhibitor proteins. Second, that NMD must be downregulated in magnitude to allow stem cells to differentiate. During the progress period, we obtained substantial evidence for both of these hypotheses. With regard to the first hypothesis, we have used genome-wide approaches to identify hundreds of messenger RNAs that are regulated by NMD in both in vivo (in mice) and in vitro (in cell lines). To determine which of these messenger mRNAs are directly degraded by NMD, we have used a variety of approaches. This work has revealed that NMD preferentially degrades messenger RNAs encoding neural differentiation inhibitors and proliferation inhibitors in neural stem cells. In contrast, very few messenger RNAs encoding pro-stem cell proteins or pro-proliferation proteins are degraded by NMD. Together this provides support for our hypothesis that NMD promotes the stem-like state by shifting the proportion of messenger RNAs in a cell towards promoting an undifferentiated, proliferative cell state. During the progress period, we have obtained considerable evidence that this hypothesis not only applies to mouse stem cells but also human embryonic stem cells. With regard to the second hypothesis, we have found that many proteins that are directly involved in the NMD pathway are downregulated upon differentiation of stem and progenitor cells. Not only are NMD proteins reduced in level, but we find that the magnitude of NMD itself is reduced. We have used a variety of molecular techniques to determine whether this NMD downregulatory response has a role in neural differentiation and found that NMD downreglation is both necessary and sufficient for this event. Such experiments have also revealed particular messenger mRNAs degraded by NMD that are crucial for the NMD downregulatory response to promote neural differentiation. During the progress period, we obtained both experimental and genome-wide data that this applies to human embryonic stem cells. Our research has implications for intellectual disability cases in humans caused by mutations in an X-linked gene essential for NMD. Patients with mutations in this gene—UPF3B—not only have intellectual disability, but also schizophrenia, autism, and attention-deficit/hyperactivity disorder. Thus, the study of NMD may provide insight into a wide spectrum of cognitive and psychological disorders. We are currently in the process of generating and characterizing induced-pluripotent stem (iPS) cells from intellectual disability patients with mutations in the UPF3B gene towards this goal.

Stem cells have the unique ability to switch from a proliferative cell state in which they reproduce themselves to a differentiated cell state that allows them to carry out the functions of fully mature cells. The stem cell field has made considerable effort to address the underlying mechanisms behind this switch. Most effort has been focused on a class of proteins that determine whether our genetic material—DNA—generates a copy of itself in the form of messenger RNA (mRNA), a process called transcription. Some factors that regulate transcription activate the production of mRNAs encoding proteins that promote the proliferative and undifferentiated cell state. Others increase the production of messenger mRNAs that encode inhibitors of differentiation and cell proliferation. While this transcription factor-centric view of stem cells explains some aspects of stem cells, it is not sufficient to explain many of their behaviors. We hypothesize that another important mechanism controlling the stem vs. differentiation switch is regulation of mRNA decay. By increasing the rate of decay of specific mRNAs, their steady-state level will be reduced, leading to lower levels of their encoded proteins being made. Likewise, decreasing mRNA decay will lead to increased mRNA level and increased protein production. We hypothesize that a particular RNA decay pathway called nonsense-mediated RNA decay (NMD) is critical for stem cell decisions. NMD recognizes specific sequence features in RNAs and then activates enzymes to degrade them. Our project revolves around two main ideas. First, that NMD promotes the stem-like state by preferentially degrading messenger RNAs that swing the balance towards the stem-like state. Second, that NMD must be downregulated in magnitude to allow stem cells to differentiate. During the latest progress period, we obtained substantial evidence for both of these hypotheses. With regard to the first hypothesis, we used genome-wide approaches to identify hundreds of mRNAs that are regulated by NMD in human embryonic stem (ES) cells. We found that one of the main classes of mRNAs regulated by NMD is those encoding signaling proteins. We examined one particular signaling pathway—called the TGFβ pathway—and found that NMD is a potent inhibitor of this pathway in human ES cells. This was of interest given that TGFβ pathway is critical for differentiation of human ES cells into endoderm, one of the three initial tissue types in mammalian embryos. We found that the magnitude of NMD is dramatically inhibited when human ES cells differentiated into endoderm, which raised the possibility that this downregulatory event allows for increased TGFβ signaling and consequent endoderm differentiation. During the progress period, we obtained several lines of evidence in support of this model. Indeed, we found that NMD downregulation is both necessary and sufficient for human ES cells to differentiate into endoderm. As further support, we found that induced pluripotent cells (iPSCs) from patients with mutations in a key NMD gene, UPF3B, have a tendency to spontaneously differentiate. UPF3B is a particularly interesting gene given that loss of UPF3B causes intellectual disability in both mice and humans (the former from our unpublished research). Patients with mutations in UPF3B also often suffer from schizophrenia, autism, and/or attention-deficit/hyperactivity disorder. Thus, the study of NMD may provide insight into a wide spectrum of cognitive and psychological disorders.

Stem cells have the unique ability to switch from a proliferative cell state in which they reproduce themselves to a differentiated cell state that allows them to carry out the functions of fully mature cells. The stem cell field has made considerable effort to address the underlying mechanisms behind this switch. Most effort has been focused on a class of proteins that determine whether our genetic material—DNA—generates a copy of itself in the form of messenger RNA (mRNA), a process called transcription. While this transcription factor-centric view of stem cells explains some aspects of stem cells, it is not sufficient to explain all of their behavior. In our grant, we proposed to test the role of another mechanism—called selective mRNA decay—that we hypothesized influences the stem vs. differentiation switch. In particular, we proposed to study nonsense-mediated RNA decay (NMD), a highly conserved RNA decay pathway that degrades specific kinds of mRNAs. One of our goals was to identify the specific mRNAs targeted for decay by NMD in human embryonic stem (ES) cells. During the final progress period, we completed this analysis, leading to a list of high-confidence NMD target mRNAs in hES cells. We found that many of these mRNAs encode proteins previously known to be important for differentiation and development. This supported the possibility that NMD acts in hES cells just as it does in mouse ES cells – to maintain them in the “stem cell state” by degrading mRNAs encoding proteins that drive differentiation. We found that another class of mRNAs degraded by NMD in hES cells is those encoding signaling proteins. This raises the possibility that NMD also acts to influence differentiation through known signaling pathways, including the FGF, WNT, TGF-beta, and BMP pathways. Another goal of our project was to determine whether NMD directly influences hES differentiation. During the last progress period we obtained additional evidence that this is the case. In brief, we found that NMD is differentially regulated during mesoderm and endoderm differentiation and that this regulation is both necessary and sufficient for human ES cells to differentiate into these two primary germ layers. As further support, we found that induced pluripotent cells (iPSCs) from patients with mutations in a key NMD gene, UPF3B, have a tendency to spontaneously differentiate into endoderm. Patients with mutations in UPF3B also often suffer from schizophrenia, autism, and/or attention-deficit/hyperactivity disorder. Thus, the study of NMD may provide insight into a wide spectrum of cognitive and psychological disorders.