Miles F. Wilkinson

Messenger RNA regulation: to translate or to degrade

Quality control of gene expression operates post‐transcriptionally at various levels in eukaryotes. Once transcribed, mRNAs associate with a host of proteins throughout their lifetime. These mRNA–protein complexes (mRNPs) undergo a series of remodeling events that are influenced by and/or influence the translation and mRNA decay machinery. In this review we discuss how a decision to translate or to degrade a cytoplasmic mRNA is reached. Nonsense‐mediated mRNA decay (NMD) and microRNA (miRNA)‐mediated mRNA silencing are provided as examples. NMD is a surveillance mechanism that detects and eliminates aberrant mRNAs whose expression would result in truncated proteins that are often deleterious to the organism. miRNA‐mediated mRNA silencing is a mechanism that ensures a given protein is expressed at a proper level to permit normal cellular function. While NMD and miRNA‐mediated mRNA silencing use different decision‐making processes to determine the fate of their targets, both are greatly influenced by mRNP dynamics. In addition, both are linked to RNA processing bodies. Possible modes involving 3′ untranslated region and its associated factors, which appear to play key roles in both processes, are discussed.

Molecular mechanism for distinct neurological phenotypes conveyed by allelic truncating mutations

The molecular mechanisms by which different mutations in the same gene can result in distinct disease phenotypes remain largely unknown. Truncating mutations of SOX10 cause either a complex neurocristopathy designated PCWH or a more restricted phenotype known as Waardenburg-Shah syndrome (WS4; OMIM 277580). Here we report that although all nonsense and frameshift mutations that cause premature termination of translation generate truncated SOX10 proteins with potent dominant-negative activity, the more severe disease phenotype, PCWH, is realized only when the mutant mRNAs escape the nonsense-mediated decay (NMD) pathway. We observe similar results for truncating mutations of MPZ that convey distinct myelinopathies. Our experiments show that triggering NMD and escaping NMD may cause distinct neurological phenotypes.

Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins

Genome-wide studies have defined cell type–specific patterns of DNA methylation that are important for regulating gene expression in both normal development and disease. However, determining the functional significance of specific methylation events remains challenging, owing to the lack of methods for removing such modifications in a targeted manner. Here we describe an approach for efficient targeted demethylation of specific CpGs in human cells using fusions of engineered transcription activator–like effector (TALE) repeat arrays and the TET1 hydroxylase catalytic domain. Using these TALE-TET1 fusions, we demonstrate that modification of critical methylated promoter CpG positions can lead to substantial increases in the expression of endogenous human genes. Our results delineate a strategy for understanding the functional significance of specific CpG methylation marks in the context of endogenous gene loci and validate programmable DNA demethylation reagents with potential utility for research and therapeutic applications.Time-course evaluation of TALE-TET1-indcued effects on HBB promoter methylation and gene expression To test whether the -266 CpG demethylation and increased HBB gene expression induced by HB-4, HB-5-, and HB-6 are stable over time, we assayed these parameters in a more extended time-course experiment. K562 cells transfected with expression plasmids encoding HB-4, HB-5, HB-6, or a control TALE-TET1 fusion targeted to the KLF4 locus were assayed for -266 CpG demethylation and HBB gene expression at 4, 7, 14, and 30 days post-transfection. HB-4, HB5, and HB-6 fusions all showed their highest levels of fold-activation at post-transfection day 4 with HBB expression steadily decreasing by day 30 to the same level observed with the control KLF4-targeted TALE-TET1 protein (Supplementary Fig. 10a). Strikingly, for all three HBBtargeted TALE-TET1 proteins, the extent of -266 CpG methylation over time in the population of transfected cells directly paralleled the fold-activation of HBB gene expression while the methylation status of another CpG at position -306 did not change significantly during the course of the experiment (Supplementary Fig. 10b).

A regulatory mechanism that detects premature nonsense codons in T-cell receptor transcripts in vivo is reversed by protein synthesis inhibitors in vitro

Gene rearrangement during the ontogeny of T- and B-cells generates an enormous repertoire of T-cell receptor (TCR) and immunoglobulin (Ig) genes. Because of the error-prone nature of this rearrangement process, two-thirds of rearranged TCR and Ig genes are expected to be out-of-frame and thus contain premature terminations codons (ptcs). We performed sequence analysis of reverse transcriptase-polymerase chain reaction products from fetal and adult thymus and found that newly transcribed TCR-β pre-mRNAs (intron-bearing) are frequently derived from ptc-bearing genes but such transcripts rarely accumulate as mature (fully spliced) TCR-β transcripts. Transfection studies in the SL12.4 T-cell line showed that the presence of a ptc in any of several TCR-β exons triggered a decrease in mRNA levels. Ptc-bearing TCR-β transcripts were selectively depressed in levels in a cell clone that contained both an in-frame and an out-of-frame gene, thus demonstrating the allelic specificity of this down-regulatory response. Protein synthesis inhibitors with different mechanism of action (anisomysin, cycloheximide, emetine, pactamycin, puromycin, and polio virus) all reversed the down-regulatory response. Ptc-bearing transcripts were induced within 0.5 h after cycloheximide treatment. The reversal by protein synthesis inhibitors was not restricted to lymphoid cells, as shown with TCR-β and β-globin constructs transfected in HeLa cells. Collectively, the data suggest that the ptc-mediated mRNA decay pathway requires an unstable protein, a ribosome, or a ribosome-like entity. Protein synthesis inhibitors may be useful tools toward elucidating the molecular mechanism of ptc-mediated mRNA decay, an enigmatic response that can occur in the nuclear fraction of mammalian cells.

A SPLICING-DEPENDENT REGULATORY MECHANISM THAT DETECTS TRANSLATION SIGNALS

Premature termination codons (PTCs) can cause the decay of mRNAs in the nuclear fraction of mammalian cells. This enigmatic nuclear response is of interest because it suggests that translation signals do not restrict their effect to the cytoplasm, where fully assembled ribosomes reside. Here we examined the molecular mechanism for this putative nuclear response by using the T‐cell receptor‐beta (TCR‐beta) gene, which acquires PTCs as a result of programmed rearrangements that occur during normal thymic ontogeny. We found that PTCs had little or no measurable effect on TCR‐beta pre‐mRNA levels, but they sharply depressed TCR‐beta mature mRNA levels in the nuclear fraction of stably transfected cells. A PTC split by an intron was able to trigger the down‐regulatory response, implying that PTC recognition occurs after an mRNA is at least partially spliced. However, intron deletion and addition studies demonstrated that a PTC must be followed by at least one functional (spliceable) intron to depress mRNA levels. One explanation for this downstream intron‐dependence is that cytoplasmic ribosomes adjacent to nuclear pores scan mRNAs still undergoing splicing as they emerge from the nucleus. We found this explanation to be unlikely because PTCs only 8 or 10 nt upstream of a terminal intron down‐regulated mRNA levels, even though this distance is too short to permit PTC recognition in the cytoplasm prior to the splicing of the downstream intron in the nucleus. Collectively, the results suggest that nonsense codon recognition may occur in the nucleus.

The Double Lives of Shuttling mRNA Binding Proteins

Several other RNA binding proteins besides hnRNPs have been identified that not only function only in the nucleus but also regulate events in the cytoplasm. These include the transcription factors Bicoid and FRGY2, and the RNA splicing regulator SXL, which have all recently been shown to regulate translation (7xGray, N.K and Wickens, M. Annu. Rev. Cell Dev. Biol. 1998; 14: 399–458Crossref | PubMed | Scopus (412)See all References, 17xMatsumoto, K, Wassarman, K, and Wolffe, A.P. EMBO J. 1998; 17: 2107–2121Crossref | PubMed | Scopus (151)See all References and references therein). Our knowledge of these RNA binding proteins suggests that many of them first complex with pre-mRNAs in the nucleus to regulate their activities and then escort mature mRNAs out to the cytoplasm to further influence their behavior. However, this appealing scenario remains unproven. Even in the case of proteins known to shuttle between the nucleus and the cytoplasm, it is not known whether these RNA binding proteins actually direct (rather than follow) mRNAs to the cytoplasm. In fact, it is not even clear whether most shuttling RNA binding proteins are bound to RNA when they traverse the nuclear pore. Instead, many RNA binding proteins may travel to the cytoplasm alone. Another unanswered question is how shuttling RNA binding proteins reach the cytoplasm. Do most of them emigrate from the nucleus, or does a selected subset set up permanent residence at their place of origin in the cytoplasm?Despite these unanswered questions, the observation that many shuttling RNA binding proteins perform duties in both the nucleus and the cytoplasm suggests the following model (Figure 1Figure 1). In the nucleus, shuttling proteins promote the assembly of a proper mRNP (mRNA/protein) complex, permitting its export to the cytoplasm. After emerging from the nucleus, these RNA binding proteins then direct the assembly or reorganization of the RNP complex to help dictate the specific outcome appropriate for a given mRNA. According to this model, the particular shuttling proteins bound to the mRNA determines where the mRNA will be localized in the cytoplasm, its rate of translation, and its rate of decay. It is possible that these shuttling proteins regulate these posttranscriptional events by interacting with microtubules, ribosomes, and degradosomes, either directly or via adaptor proteins.Figure 1Model for How Shuttling mRNA Binding Proteins Regulate Events in Both the Nucleus and the CytoplasmPABP, poly(A) binding protein; eIF4F, eukaryotic initiation factor 4F.View Large Image | View Hi-Res Image | Download PowerPoint SlideThe notion that nuclear proteins travel with mRNAs to the cytoplasm to regulate mRNA function provides an explanation for a series of observations that the nuclear history of an mRNA can affect its cytoplasmic fate. For example, some genes have been shown to require introns for efficient translation of their spliced mRNA products in the cytoplasm (Matsumoto et al. 1998xMatsumoto, K, Wassarman, K, and Wolffe, A.P. EMBO J. 1998; 17: 2107–2121Crossref | PubMed | Scopus (151)See all ReferencesMatsumoto et al. 1998). In addition, as mentioned earlier, introns are necessary to engage the mammalian NMD RNA surveillance pathway that detects nonsense codons by a mechanism with features of the cytoplasmic translational machinery. To explain these observations, it has been hypothesized that RNA binding proteins exist that regulate intron-dependent events in the nucleus and then go on to the cytoplasm to regulate subsequent events. Future studies will be required to test this theory and, if it is true, to identify the specific RNA binding shuttling proteins involved and how they communicate with each other.How RNA binding shuttling proteins themselves are regulated also remains to be determined. Is their distribution between the nucleus and the cytoplasm modulated by environmental cues? Do posttranslational events, such as phosphorylation and methylation, alter their ability to control gene expression events in the nucleus or the cytoplasm? The answer appears to be yes, as a recent study showed that the p38 stress-activated mitogen-activated kinase alters the nucleocytoplasmic distribution of hnRNP A1 in response to osmotic stress, leading to changes of alternative splicing (van der Houven van Oordt et al. 2000xvan der Houven van Oordt, W, Diaz-Meco, M.T, Lozano, J, Krainer, A.R, Moscat, J, and Caceres, J.F. J. Cell Biol. 2000; 149: 307–316Crossref | PubMed | Scopus (224)See all Referencesvan der Houven van Oordt et al. 2000). As stress-activated signaling pathways are fundamental to the life and death of cells, they could play a key role in modulating the functional linkage between mRNA metabolism in the cytoplasmic and nuclear compartments under both physiological conditions and stress responses. The answers to these questions will ultimately tell us much about how communication between the nuclear and cytoplasmic compartments is orchestrated.‡To whom correspondence should be addressed (e-mail: ann-bin.shyu@uth.tmc.edu).

NONSENSE SURVEILLANCE IN LYMPHOCYTES

We would like to thank the following people for providing comments on this minireview: Thomas Cooper, Gilbert Cote, Maureen Goode, Hector Martinez-Valdez, Thomas Perrin, David Roth, Phillip Sharp, Marc Shulman, Ann-Bin Shyu, Ursula Storb, and Carrie Valentine.

Identification of a MicroRNA that Activates Gene Expression by Repressing Nonsense-Mediated RNA Decay

Nonsense-mediated decay (NMD) degrades both normal and aberrant transcripts harboring stop codons in particular contexts. Mutations that perturb NMD cause neurological disorders in humans, suggesting that NMD has roles in the brain. Here, we identify a brain-specific microRNA-miR-128-that represses NMD and thereby controls batteries of transcripts in neural cells. miR-128 represses NMD by targeting the RNA helicase UPF1 and the exon-junction complex core component MLN51. The ability of miR-128 to regulate NMD is a conserved response occurring in frogs, chickens, and mammals. miR-128 levels are dramatically increased in differentiating neuronal cells and during brain development, leading to repressed NMD and upregulation of mRNAs normally targeted for decay by NMD; overrepresented are those encoding proteins controlling neuron development and function. Together, these results suggest the existence of a conserved RNA circuit linking the microRNA and NMD pathways that induces cell type-specific transcripts during development.

Rhox: A New Homeobox Gene Cluster

Homeobox genes encode transcription factors notable for their ability to regulate embryogenesis. Here, we report the discovery of a cluster of 12 related homeobox genes on the X chromosome expressed in male and female reproductive tissues in adult mice. These reproductive homeobox on the X chromosome (Rhox) genes are expressed in a cell type-specific manner; several are hormonally regulated, and their expression pattern during postnatal testis development corresponds to their chromosomal position. Most of the Rhox genes are expressed in Sertoli cells, the nurse cells in direct contact with developing male germ cells, suggesting that they regulate the expression of somatic-cell gene products critical for germ cell development. In support of this, targeted disruption of Rhox5 increased male germ cell apoptosis and reduced sperm production, sperm motility, and fertility. Identification of this family of homeobox genes provides an opportunity to study colinear gene regulation and the transcriptional control of reproduction.

An alternative branch of the nonsense-mediated decay pathway

The T‐cell receptor (TCR) locus undergoes programmed rearrangements that frequently generate premature termination codons (PTCs). The PTC‐bearing transcripts derived from such nonproductively rearranged genes are dramatically downregulated by the nonsense‐mediated decay (NMD) pathway. Here, we show that depletion of the NMD factor UPF3b does not impair TCRβ NMD, thereby distinguishing it from classical NMD. Depletion of the related factor UPF3a, by itself or in combination with UPF3b, also has no effect on TCRβ NMD. Mapping experiments revealed the identity of TCRβ sequences that elicit a switch to UPF3b dependence. This regulation is not a peculiarity of TCRβ, as we identified many wild‐type genes, including one essential for NMD, that transcribe NMD‐targeted mRNAs whose downregulation is little or not affected by UPF3a and UPF3b depletion. We propose that we have uncovered an alternative branch of the NMD pathway that not only degrades aberrant mRNAs but also regulates normal mRNAs, including one that participates in a negative feedback loop controlling the magnitude of NMD.