Lulu Huang

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.

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.

RNA homeostasis governed by cell type-specific and branched feedback loops acting on NMD.

Nonsense-mediated mRNA decay (NMD) is a conserved RNA decay pathway that degrades aberrant mRNAs and directly regulates many normal mRNAs. This dual role for NMD raises the possibility that its magnitude is buffered to prevent the potentially catastrophic alterations in gene expression that would otherwise occur if NMD were perturbed by environmental or genetic insults. In support of this, here we report the existence of a negative feedback regulatory network that directly acts on seven NMD factors. Feedback regulation is conferred by different branches of the NMD pathway in a cell type-specific and developmentally regulated manner. We identify feedback-regulated NMD factors that are rate limiting for NMD and demonstrate that reversal of feedback regulation in response to NMD perturbation is crucial for maintaining NMD. Together, our results suggest the existence of an intricate feedback network that maintains both RNA surveillance and the homeostasis of normal gene expression in mammalian cells.

Regulation of nonsense-mediated mRNA decay

Nonsense‐mediated mRNA decay (NMD) is a highly conserved pathway that was originally identified as a RNA surveillance mechanism that degrades aberrant mRNAs harboring premature termination (nonsense) codons. Recently, it was discovered that NMD also regulates normal gene expression. Genome‐wide studies showed that ablation of NMD alters the expression of ∼10% of transcripts in a wide variety of eukaryotes. In general, NMD specifically targets normal transcripts that harbor a stop codon in a premature context. The finding that NMD regulates normal gene expression raises the possibility that NMD itself is subject to regulation. Indeed, recent studies have shown that NMD efficiency varies in different cell types and tissues. NMD is also subject to developmental control in both higher and lower eukaryotic species. Molecular mechanisms have been defined—including those involving microRNAs and other RNA decay pathways—that regulate the magnitude of NMD in some developmental settings. This developmental regulation of NMD appears to have physiological roles, at least in some model systems. In addition to mechanisms that modulate the efficiency of NMD, mechanisms have recently been identified that serve the opposite purpose: to maintain the efficiency of NMD in the face of insults. This ‘buffering’ is achieved by feedback networks that serve to regulate the stability of NMD factors. The discovery of NMD homeostasis and NMD regulatory mechanisms has important implications for how NMD acts in biological processes and how its magnitude could potentially be manipulated for clinical benefit. WIREs RNA 2012. doi: 10.1002/wrna.1137

A UPF3-mediated regulatory switch that maintains RNA surveillance

Nonsense-mediated decay (NMD) is an RNA decay pathway that downregulates aberrant mRNAs and a subset of normal mRNAs. The regulation of NMD is poorly understood. Here we identify a regulatory mechanism acting on two related UPF (up-frameshift) factors crucial for NMD: UPF3A and UPF3B. This regulatory mechanism, which reduces the level of UPF3A in response to the presence of UPF3B, is relieved in individuals harboring UPF3B mutations, leading to strongly increased steady-state levels of UPF3A. UPF3A compensates for the loss of UPF3B by regulating several NMD target transcripts, but it can also impair NMD, as it competes with the stronger NMD activator UPF3B for binding to the essential NMD factor UPF2. This deleterious effect of UPF3A protein is prevented by its destabilization using a conserved UPF3B-dependent mechanism. Together, our results suggest that UPF3A levels are tightly regulated by a post-transcriptional switch to maintain appropriate levels of NMD substrates in cells containing different levels of UPF3B.

The unfolded protein response is shaped by the NMD pathway.

Endoplasmic reticulum (ER) stress induces the unfolded protein response (UPR), an essential adaptive intracellular pathway that relieves the stress. Although the UPR is an evolutionarily conserved and beneficial pathway, its chronic activation contributes to the pathogenesis of a wide variety of human disorders. The fidelity of UPR activation must thus be tightly regulated to prevent inappropriate signaling. The nonsense‐mediated RNA decay (NMD) pathway has long been known to function in RNA quality control, rapidly degrading aberrant mRNAs, and has been suggested to regulate subsets of normal mRNAs. Here, we report that the NMD pathway regulates the UPR. NMD increases the threshold for triggering the UPR in vitro and in vivo, thereby preventing UPR activation in response to normally innocuous levels of ER stress. NMD also promotes the timely termination of the UPR. We demonstrate that NMD directly targets the mRNAs encoding several UPR components, including the highly conserved UPR sensor, IRE1α, whose NMD‐dependent degradation partly underpins this process. Our work not only sheds light on UPR regulation, but demonstrates the physiological relevance of NMDs ability to regulate normal mRNAs.

A Upf3b-mutant mouse model with behavioral and neurogenesis defects.

Nonsense-mediated RNA decay (NMD) is a highly conserved and selective RNA degradation pathway that acts on RNAs terminating their reading frames in specific contexts. NMD is regulated in a tissue-specific and developmentally controlled manner, raising the possibility that it influences developmental events. Indeed, loss or depletion of NMD factors have been shown to disrupt developmental events in organisms spanning the phylogenetic scale. In humans, mutations in the NMD factor gene, UPF3B, cause intellectual disability (ID) and are strongly associated with autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD) and schizophrenia (SCZ). Here, we report the generation and characterization of mice harboring a null Upf3b allele. These Upf3b-null mice exhibit deficits in fear-conditioned learning, but not spatial learning. Upf3b-null mice also have a profound defect in prepulse inhibition (PPI), a measure of sensorimotor gating commonly deficient in individuals with SCZ and other brain disorders. Consistent with both their PPI and learning defects, cortical pyramidal neurons from Upf3b-null mice display deficient dendritic spine maturation in vivo. In addition, neural stem cells from Upf3b-null mice have impaired ability to undergo differentiation and require prolonged culture to give rise to functional neurons with electrical activity. RNA sequencing (RNAseq) analysis of the frontal cortex identified UPF3B-regulated RNAs, including direct NMD target transcripts encoding proteins with known functions in neural differentiation, maturation and disease. We suggest Upf3b-null mice serve as a novel model system to decipher cellular and molecular defects underlying ID and neurodevelopmental disorders.