Regulated dicing of pre-mir-144 via reshaping of its terminal loop.

Although the route to generate microRNAs (miRNAs) is often depicted as a linear series of sequential and constitutive cleavages, we now appreciate multiple alternative pathways as well as diverse strategies to modulate their processing and function. Here, we identify an unusually profound regulatory role of conserved loop sequences in vertebrate pre-mir-144, which are essential for its cleavage by the Dicer RNase III enzyme in human and zebrafish models. Our data indicate that pre-mir-144 dicing is positively regulated via its terminal loop, and involves the ILF3 complex (NF90 and its partner NF45/ILF2). We provide further evidence that this regulatory switch involves reshaping of the pre-mir-144 apical loop into a structure that is appropriate for Dicer cleavage. In light of our recent findings that mir-144 promotes the nuclear biogenesis of its neighbor mir-451, these data extend the complex hierarchy of nuclear and cytoplasmic regulatory events that can control the maturation of clustered miRNAs.

The Clp1 R140H mutation alters tRNA metabolism and mRNA 3' processing in mouse models of pontocerebellar hypoplasia.

Homozygous mutation of the RNA kinase CLP1 (cleavage factor polyribonucleotide kinase subunit 1) causes pontocerebellar hypoplasia type 10 (PCH10), a pediatric neurodegenerative disease. CLP1 is associated with the transfer RNA (tRNA) splicing endonuclease complex and the cleavage and polyadenylation machinery, but its function remains unclear. We generated two mouse models of PCH10: one homozygous for the disease-associated Clp1 mutation, R140H, and one heterozygous for this mutation and a null allele. Both models exhibit loss of lower motor neurons and neurons of the deep cerebellar nuclei. To explore whether Clp1 mutation impacts tRNA splicing, we profiled the products of intron-containing tRNA genes. While mature tRNAs were expressed at normal levels in mutant mice, numerous other products of intron-containing tRNA genes were dysregulated, with pre-tRNAs, introns, and certain tRNA fragments up-regulated, and other fragments down-regulated. However, the spatiotemporal patterns of dysregulation do not correlate with pathogenicity for most altered tRNA products. To elucidate the effect of Clp1 mutation on precursor messenger RNA (pre-mRNA) cleavage, we analyzed poly(A) site (PAS) usage and gene expression in Clp1R140H/- spinal cord. PAS usage was shifted from proximal to distal sites in the mutant mouse, particularly in short and closely spaced genes. Many such genes were also expressed at lower levels in the Clp1R140H/- mouse, possibly as a result of impaired transcript maturation. These findings are consistent with the hypothesis that select genes are particularly dependent upon CLP1 for proper pre-mRNA cleavage, suggesting that impaired mRNA 3' processing may contribute to pathogenesis in PCH10.

SARNAclust: Semi-automatic detection of RNA protein binding motifs from immunoprecipitation data.

RNA-protein binding is critical to gene regulation, controlling fundamental processes including splicing, translation, localization and stability, and aberrant RNA-protein interactions are known to play a role in a wide variety of diseases. However, molecular understanding of RNA-protein interactions remains limited; in particular, identification of RNA motifs that bind proteins has long been challenging, especially when such motifs depend on both sequence and structure. Moreover, although RNA binding proteins (RBPs) often contain more than one binding domain, algorithms capable of identifying more than one binding motif simultaneously have not been developed. In this paper we present a novel pipeline to determine binding peaks in crosslinking immunoprecipitation (CLIP) data, to discover multiple possible RNA sequence/structure motifs among them, and to experimentally validate such motifs. At the core is a new semi-automatic algorithm SARNAclust, the first unsupervised method to identify and deconvolve multiple sequence/structure motifs simultaneously. SARNAclust computes similarity between sequence/structure objects using a graph kernel, providing the ability to isolate the impact of specific features through the bulge graph formalism. Application of SARNAclust to synthetic data shows its capability of clustering 5 motifs at once with a V-measure value of over 0.95, while GraphClust achieves only a V-measure of 0.083 and RNAcontext cannot detect any of the motifs. When applied to existing eCLIP sets, SARNAclust finds known motifs for SLBP and HNRNPC and novel motifs for several other RBPs such as AGGF1, AKAP8L and ILF3. We demonstrate an experimental validation protocol, a targeted Bind-n-Seq-like high-throughput sequencing approach that relies on RNA inverse folding for oligo pool design, that can validate the components within the SLBP motif. Finally, we use this protocol to experimentally interrogate the SARNAclust motif predictions for protein ILF3. Our results support a newly identified partially double-stranded UUUUUGAGA motif similar to that known for the splicing factor HNRNPC.

Cancer stem cells, not bulk tumor cells, determine mechanisms of resistance to SMO inhibitors.

The emergence of treatment resistance significantly reduces the clinical utility of many effective targeted therapies. Although both genetic and epigenetic mechanisms of drug resistance have been reported, whether these mechanisms are stochastically selected in individual tumors or governed by a predictable underlying principle is unknown. Here, we report that the dependence of cancer stem cells (CSCs), not bulk tumor cells, on the targeted pathway determines the molecular mechanism of resistance in individual tumors. Using both spontaneous and transplantable mouse models of sonic hedgehog (SHH) medulloblastoma (MB) treated with an SHH/Smoothened inhibitor, sonidegib/LDE225, we show that genetic-based resistance occurs only in tumors that contain SHH-dependent CSCs (SD-CSCs). In contrast, SHH MBs containing SHH-dependent bulk tumor cells but SHH-independent CSCs (SI-CSCs) acquire resistance through epigenetic reprogramming. Mechanistically, elevated proteasome activity in SMOi-resistant SI-CSC MBs alters the tumor cell maturation trajectory through enhanced degradation of specific epigenetic regulators, including histone acetylation machinery components, resulting in global reductions in H3K9Ac, H3K14Ac, H3K56Ac, H4K5Ac, and H4K8Ac marks and gene expression changes. These results provide new insights into how selective pressure on distinct tumor cell populations contributes to different mechanisms of resistance to targeted therapies. This insight provides a new conceptual framework to understand responses and resistance to SMOis and other targeted therapies.

Defects in translation-dependent quality control pathways lead to convergent molecular and neurodevelopmental pathology.

Translation-dependent quality control pathways such as no-go decay (NGD), non-stop decay (NSD), and nonsense-mediated decay (NMD) govern protein synthesis and proteostasis by resolving non-translating ribosomes and preventing the production of potentially toxic peptides derived from faulty and aberrant mRNAs. However, how translation is altered and the in vivo defects that arise in the absence of these pathways are poorly understood. Here, we show that the NGD/NSD factors Pelo and Hbs1l are critical in mice for cerebellar neurogenesis but expendable for survival of these neurons after development. Analysis of mutant mouse embryonic fibroblasts revealed translational pauses, alteration of signaling pathways, and translational reprogramming. Similar effects on signaling pathways, including mTOR activation, the translatome and mouse cerebellar development were observed upon deletion of the NMD factor Upf2. Our data reveal that these quality control pathways that function to mitigate errors at distinct steps in translation can evoke similar cellular responses.

Expression of the Neuronal tRNA n-Tr20 Regulates Synaptic Transmission and Seizure Susceptibility.

The mammalian genome has hundreds of nuclear-encoded tRNAs, but the contribution of individual tRNA genes to cellular and organismal function remains unknown. Here, we demonstrate that mutations in a neuronally enriched arginine tRNA, n-Tr20, increased seizure threshold and altered synaptic transmission. n-Tr20 expression also modulated seizures caused by an epilepsy-linked mutation in Gabrg2, a gene encoding a GABAA receptor subunit. Loss of n-Tr20 altered translation initiation by activating the integrated stress response and suppressing mTOR signaling, the latter of which may contribute to altered neurotransmission in mutant mice. Deletion of a highly expressed isoleucine tRNA similarly altered these signaling pathways in the brain, suggesting that regulation of translation initiation is a conserved response to tRNA loss. Our data indicate that loss of a single member of a tRNA family results in multiple cellular phenotypes, highlighting the disease-causing potential of tRNA mutations.

GTPBP1 resolves paused ribosomes to maintain neuronal homeostasis.

Ribosome-associated quality control pathways respond to defects in translational elongation to recycle arrested ribosomes and degrade aberrant polypeptides and mRNAs. Loss of a tRNA gene leads to ribosomal pausing that is resolved by the translational GTPase GTPBP2, and in its absence causes neuron death. Here, we show that loss of the homologous protein GTPBP1 during tRNA deficiency in the mouse brain also leads to codon-specific ribosome pausing and neurodegeneration, suggesting that these non-redundant GTPases function in the same pathway to mitigate ribosome pausing. As observed in Gtpbp2-/- mice (Ishimura et al., 2016), GCN2-mediated activation of the integrated stress response (ISR) was apparent in the Gtpbp1-/- brain. We observed decreased mTORC1 signaling which increased neuronal death, whereas ISR activation was neuroprotective. Our data demonstrate that GTPBP1 functions as an important quality control mechanism during translation elongation and suggest that translational signaling pathways intricately interact to regulate neuronal homeostasis during defective elongation.

Vex-seq: high-throughput identification of the impact of genetic variation on pre-mRNA splicing efficiency.

Understanding the functional impact of genomic variants is a major goal of modern genetics and personalized medicine. Although many synonymous and non-coding variants act through altering the efficiency of pre-mRNA splicing, it is difficult to predict how these variants impact pre-mRNA splicing. Here, we describe a massively parallel approach we use to test the impact on pre-mRNA splicing of 2059 human genetic variants spanning 110 alternative exons. This method, called variant exon sequencing (Vex-seq), yields data that reinforce known mechanisms of pre-mRNA splicing, identifies variants that impact pre-mRNA splicing, and will be useful for increasing our understanding of genome function.