Co-phase separation of Y14 and RNA in vitro and its implication for DNA repair.

The multifunctional RNA recognition motif-containing protein Y14/RBM8A participates in mRNA metabolism and is essential for the efficient repair of DNA double-strand breaks (DSBs). Y14 contains highly charged, low-complexity sequences in both the amino- and carboxy-terminal domains. The feature of charge segregation suggests that Y14 may undergo liquid-liquid phase separation (LLPS). Recombinant Y14 formed phase-separated droplets, which were sensitive to pH and salt concentration. Domain mapping suggested that LLPS of Y14 involves multivalent electrostatic interactions and is partly determined by the net charge of its low-complexity regions. Phospho-mimicry of the carboxy-terminal arginine-serine dipeptides of Y14 suppressed phase separation. Moreover, RNA could phase separate into Y14 droplets and modulate Y14 LLPS in a concentration-dependent manner. Finally, the capacity of Y14 in LLPS and coacervation with RNA in vitro correlated with its activity in DSB repair. These results reveal a molecular rule for LLPS of Y14 in vitro and an implication for its co-condensation with RNA in genome stability.

The pioneer round of translation: features and functions.

In mammalian cells, newly synthesized mRNAs undergo a pioneer round of translation that is important for mRNA quality control. Following maturation of messenger ribonucleoprotein particles during and after the pioneer round, steady-state cycles of mRNA translation generate most of the cell's proteins. Translation factors, RNA-binding proteins, and targets of signaling pathways that are particular to newly synthesized mRNAs regulate critical functions of the pioneer round.

LncRNA HOTAIRM1 functions in DNA double-strand break repair via its association with DNA repair and mRNA surveillance factors.

The eukaryotic exon junction complex component Y14 participates in double-strand break (DSB) repair via its RNA-dependent interaction with the non-homologous end-joining (NHEJ) complex. Using immunoprecipitation-RNA-seq, we identified a set of Y14-associated long non-coding RNAs (lncRNAs). The lncRNA HOTAIRM1 serves as a strong candidate that mediates the interaction between Y14 and the NHEJ complex. HOTAIRM1 localized to near ultraviolet laser-induced DNA damage sites. Depletion of HOTAIRM1 delayed the recruitment of DNA damage response and repair factors to DNA lesions and compromised the efficiency of NHEJ-mediated DSB repair. Identification of the HOTAIRM1 interactome revealed a large set of RNA processing factors including mRNA surveillance factors. The surveillance factors Upf1 and SMG6 localized to DNA damage sites in a HOTAIRM1-dependent manner. Depletion of Upf1 or SMG6 increased the level of DSB-induced non-coding transcripts at damaged sites, indicating a pivotal role for Upf1/SMG6-mediated RNA degradation in DNA repair. We conclude that HOTAIRM1 serves as an assembly scaffold for both DNA repair and mRNA surveillance factors that act in concert to repair DSBs.

p53 Activation in Genetic Disorders: Different Routes to the Same Destination.

The tumor suppressor p53 is critical for preventing neoplastic transformation and tumor progression. Inappropriate activation of p53, however, has been observed in a number of human inherited disorders that most often affect development of the brain, craniofacial region, limb skeleton, and hematopoietic system. Genes related to these developmental disorders are essentially involved in transcriptional regulation/chromatin remodeling, rRNA metabolism, DNA damage-repair pathways, telomere maintenance, and centrosome biogenesis. Perturbation of these activities or cellular processes may result in p53 accumulation in cell cultures, animal models, and perhaps humans as well. Mouse models of several p53 activation-associated disorders essentially recapitulate human traits, and inactivation of p53 in these models can alleviate disorder-related phenotypes. In the present review, we focus on how dysfunction of the aforementioned biological processes causes developmental defects via excessive p53 activation. Notably, several disease-related genes exert a pleiotropic effect on those cellular processes, which may modulate the magnitude of p53 activation and establish or disrupt regulatory loops. Finally, we discuss potential therapeutic strategies for genetic disorders associated with p53 misactivation.

uORF-mediated translational control: recently elucidated mechanisms and implications in cancer.

Protein synthesis is tightly regulated, and its dysregulation can contribute to the pathology of various diseases, including cancer. Increased or selective translation of mRNAs can promote cancer cell proliferation, metastasis and tumor expansion. Translational control is one of the most important means for cells to quickly adapt to environmental stresses. Adaptive translation involves various alternative mechanisms of translation initiation. Upstream open reading frames (uORFs) serve as a major regulator of stress-responsive translational control. Since recent advances in omics technologies including ribo-seq have expanded our knowledge of translation, we discuss emerging mechanisms for uORF-mediated translation regulation and its impact on cancer cell biology. A better understanding of dysregulated translational control of uORFs in cancer would facilitate the development of new strategies for cancer therapy.

DDX3 Modulates Neurite Development via Translationally Activating an RNA Regulon Involved in Rac1 Activation.

UNLABELLED: The RNA helicase DDX3 is a component of neuronal granules, and its gene mutations are linked to intellectual disability (ID). Here we demonstrate that DDX3 depletion in neurons impairs neurite development by downregulating Rac1 level and activation. Moreover, DDX3 activates the translation of functionally coherent mRNAs involved in Rac1 activation including Rac1 Among the DDX3 regulon, Prkaca encodes the catalytic subunit of PKA, a potential activator of Rac1 in neurons. DDX3-modulated PKAcα and Rac1 expression tunes the strength of PKA-Rac1 signaling and thereby contributes to neurite outgrowth and dendritic spine formation. Inhibition of DDX3 activity or expression in neonatal mice impaired dendritic outgrowth and spine formation of hippocampal neurons, echoing neuronal deficits underling DDX3 mutation-associated ID. Finally, we provide evidence that DDX3 activates local protein synthesis through a 5' UTR-dependent mechanism in neurons. The novel DDX3 regulon may conduct a spatial and temporal control of Rac1 signaling to regulate neurite development. SIGNIFICANCE STATEMENT: DDX3X mutations are linked to intellectual disability (ID). We provide first evidence that DDX3 is required for neurite outgrowth and dendritic spine formation in vitro and in vivo We identified a DDX3 regulon constituting functionally cohesive mRNAs involved in Rac1 signaling, which contributes to DDX3-modulated neurite development. Inhibition or ablation of DDX3 in vivo shortened neurite lengths and impaired dendritic spine formation in hippocampal neurons, reflecting the prevalence of ID-associated DDX3X mutations in the helicase domain. Mechanistically, DDX3 activates local protein synthesis of mRNAs sharing similar 5' UTR structures and therefore controls Rac1 signaling strength in neurites.

A Point Mutation in the Exon Junction Complex Factor Y14 Disrupts Its Function in mRNA Cap Binding and Translation Enhancement.

Eukaryotic mRNA biogenesis involves a series of interconnected steps mediated by RNA-binding proteins. The exon junction complex core protein Y14 is required for nonsense-mediated mRNA decay (NMD) and promotes translation. Moreover, Y14 binds the cap structure of mRNAs and inhibits the activity of the decapping enzyme Dcp2. In this report, we show that an evolutionarily conserved tryptophan residue (Trp-73) of Y14 is critical for its binding to the mRNA cap structure. A Trp-73 mutant (W73V) bound weakly to mRNAs and failed to protect them from degradation. However, this mutant could still interact with the NMD and mRNA degradation factors and retained partial NMD activity. In addition, we found that the W73V mutant could not interact with translation initiation factors. Overexpression of W73V suppressed reporter mRNA translation in vitro and in vivo and reduced the level of a set of nascent proteins. These results reveal a residue of Y14 that confers cap-binding activity and is essential for Y14-mediated enhancement of translation. Finally, we demonstrated that Y14 may selectively and differentially modulate protein biosynthesis.

Elevated SRPK1 lessens apoptosis in breast cancer cells through RBM4-regulated splicing events.

Imbalanced splicing of premessenger RNA is typical of tumorous malignancies, and the regulatory mechanisms involved in several tumorigenesis-associated splicing events are identified. Elevated expression of serine-arginine protein kinase 1 (SRPK1) may participate in the pathway responsible for the dysregulation of splicing events in malignant tumor cells. In this study, we observed a correlation between the cytoplasmic accumulation of RNA-binding motif protein 4 (RBM4) and up-regulated SRPK1 in breast cancer cells. The production of the IR-B and MCL-1S transcripts was induced separately by the overexpression of RBM4 and SRPK1 gene silencing. Overexpressed RBM4 simultaneously bound to the CU-rich elements within the MCL-1 exon2 and the downstream intron, which subsequently facilitated the exclusion of the regulated exon. Breast cancer cells are deprived of apoptotic resistance through the RBM4-mediated up-regulation of the IR-B and MCL-1S transcripts. These findings suggest that the splicing events regulated by the SRPK1-RMB4 network may contribute to tumorigenesis through altered sensitivity to apoptotic signals in breast cancer cells.

TRAP150 activates splicing in composite terminal exons.

The spliceosomal factor TRAP150 is essential for pre-mRNA splicing in vivo and, when overexpressed, it enhances splicing efficiency. In this study, we found that TRAP150 interacted with the cleavage and polyadenylation specificity factor (CPSF) and co-fractionated with CPSF and RNA polymerase II. Moreover, TRAP150 preferentially associated with the U1 small ribonucleoprotein (snRNP). However, our data do not support a role for TRAP150 in alternative 5' splice site or exon selection or in alternative polyadenylation. Because U1 snRNP participates in premature cleavage and polyadenylation (PCPA), we tested whether TRAP150 is a cofactor in the control of PCPA. Although TRAP150 depletion had no significant effect on PCPA, overexpression of TRAP150 forced activation of a cryptic 3' splice site, yielding spliced PCPA transcripts. Mechanistic studies showed that TRAP150-activated splicing occurred in composite but not authentic terminal exons, and such an activity was enhanced by debilitation of U1 snRNP or interference with transcription elongation or termination. Together, these results indicate that TRAP150 provides an additional layer of PCPA regulation, through which it may increase the diversity of abortive RNA transcripts under conditions of compromised gene expression.

Alanine repeats influence protein localization in splicing speckles and paraspeckles.

Mammalian splicing regulatory protein RNA-binding motif protein 4 (RBM4) has an alanine repeat-containing C-terminal domain (CAD) that confers both nuclear- and splicing speckle-targeting activities. Alanine-repeat expansion has pathological potential. Here we show that the alanine-repeat tracts influence the subnuclear targeting properties of the RBM4 CAD in cultured human cells. Notably, truncation of the alanine tracts redistributed a portion of RBM4 to paraspeckles. The alanine-deficient CAD was sufficient for paraspeckle targeting. On the other hand, alanine-repeat expansion reduced the mobility of RBM4 and impaired its splicing activity. We further took advantage of the putative coactivator activator (CoAA)-RBM4 conjoined splicing factor, CoAZ, to investigate the function of the CAD in subnuclear targeting. Transiently expressed CoAZ formed discrete nuclear foci that emerged and subsequently separated-fully or partially-from paraspeckles. Alanine-repeat expansion appeared to prevent CoAZ separation from paraspeckles, resulting in their complete colocalization. CoAZ foci were dynamic but, unlike paraspeckles, were resistant to RNase treatment. Our results indicate that the alanine-rich CAD, in conjunction with its conjoined RNA-binding domain(s), differentially influences the subnuclear localization and biogenesis of RBM4 and CoAZ.

Emerging role for RNA binding motif protein 4 in the development of brown adipocytes.

RNA-binding motif protein 4 (RBM4) reportedly reprograms the tissue-specific splicing network which modulates the development of muscles and pancreatic ß-islets. Herein, we report that Rbm4a(-/-) mice exhibited hyperlipidemia accompanied with reduced mass of interscapular brown adipose tissue (iBAT). Elevated RBM4a led to the isoform shift of IR, Ppar-γ, and Pref-1 genes which play pivotal roles in the different stages of adipogenesis. Overexpression of RBM4a enhanced the mitochondrial activity of brown adipocyte-like lineage in the presence of uncoupling agent. RBM4a-ablated adipocytes inversely exhibited impaired development and inefficient energy expenditure. Intriguingly, overexpressed RBM4a induced the expression of brown adipocyte-specific factors (Prdm16 and Bmp7) in white adipocyte-like lineage, which suggested the potential action of RBM4a on the white-to-brown trans-differentiation of adipocytes. In differentiating adipocytes, RBM4a constituted a feed-forward circuit through autoregulating the splicing pattern of its own transcript. Based on these results, we propose the emerging role of RBM4 in regulating the adipocyte-specific splicing events and transcription cascade, which subsequently facilitate the development and function of brown adipocyte-like cells.

BCAS2 is essential for Drosophila viability and functions in pre-mRNA splicing.

Here, we show that dBCAS2 (CG4980, human Breast Carcinoma Amplified Sequence 2 ortholog) is essential for the viability of Drosophila melanogaster. We find that ubiquitous or tissue-specific depletion of dBCAS2 leads to larval lethality, wing deformities, impaired splicing, and apoptosis. More importantly, overexpression of hBCAS2 rescues these defects. Furthermore, the C-terminal coiled-coil domain of hBCAS2 binds directly to CDC5L and recruits hPrp19/PLRG1 to form a core complex for splicing in mammalian cells and can partially restore wing damage induced by knocking down dBCAS2 in flies. In summary, Drosophila and human BCAS2 share a similar function in RNA splicing, which affects cell viability.

DDX3 regulates cancer immune surveillance via 3' UTR-mediated cell-surface expression of PD-L1.

Programmed death-1 (PD-1)/PD ligand-1 (PD-L1)-mediated immune escape contributes to cancer development and has been targeted as an anti-cancer strategy. Here, we show that inhibition of the RNA helicase DDX3 increased CD8+ T cell infiltration in syngeneic oral squamous cell carcinoma tumors. DDX3 knockdown compromised interferon-γ-induced PD-L1 expression and, in particular, reduced the level of cell-surface PD-L1. DDX3 promoted surface PD-L1 expression by recruiting the adaptor protein 2 (AP2) complex to the 3' UTR of PD-L1 mRNA. DDX3 depletion or 3' UTR truncation increased the binding of the coatomer protein complexes to PD-L1, leading to its intracellular accumulation. Therefore, this 3' UTR-dependent mechanism may counteract cellular negative effects on surface trafficking of PD-L1. Finally, pharmaceutic disruption of DDX3's interaction with AP2 reduced surface PD-L1 expression, supporting that the DDX3-AP2 pathway routes PD-L1 to the cell surface. Targeting DDX3 to modulate surface trafficking of immune checkpoint proteins may provide a potential strategy for cancer immunotherapy.

Coupling pre-mRNA processing to transcription on the RNA factory assembly line.

It has been well-documented that nuclear processing of primary transcripts of RNA polymerase II occurs co-transcriptionally and is functionally coupled to transcription. Moreover, increasing evidence indicates that transcription influences pre-mRNA splicing and even several post-splicing RNA processing events. In this review, we discuss the issues of how RNA polymerase II modulates co-transcriptional RNA processing events via its carboxyl terminal domain, and the protein domains involved in coupling of transcription and RNA processing events. In addition, we describe how transcription influences the expression or stability of mRNAs through the formation of distinct mRNP complexes. Finally, we delineate emerging findings that chromatin modifications function in the regulation of RNA processing steps, especially splicing, in addition to transcription. Overall, we provide a comprehensive view that transcription could integrate different control systems, from epigenetic to post-transcriptional control, for efficient gene expression.

Activation of TrkB signaling mitigates cerebellar anomalies caused by Rbm4-Bdnf deficiency.

A molecular and functional link between neurotrophin signaling and cerebellar foliation is lacking. Here we show that constitutive knockout of two homologous genes encoding the RNA binding protein RBM4 results in foliation defects at cerebellar lobules VI-VII and delayed motor learning in mice. Moreover, the features of Rbm4 double knockout (dKO), including impaired differentiation of cerebellar granule cells and dendritic arborization of Purkinje cells, are reminiscent of neurotrophin deficiency. Loss of RBM4 indeed reduced brain-derived neurotrophic factor (BDNF). RBM4 promoted the expression of BDNF and full-length TrkB, implicating RBM4 in efficient BDNF-TrkB signaling. Finally, prenatal supplementation with 7,8-dihydroxyflavone, a TrkB agonist, restored granule cell differentiation, Purkinje cell dendritic complexity and foliation-the intercrural fissure in particular-in the neonatal cerebellum of Rbm4dKO mice, which also showed improved motor learning in adulthood. This study provides evidence that prenatal activation of TrkB signaling ameliorates cerebellar malformation caused by BDNF deficiency.

The exon junction complex component Y14 modulates the activity of the methylosome in biogenesis of spliceosomal small nuclear ribonucleoproteins.

The RNA-binding protein Y14 heterodimerizes with Mago as the core of the exon junction complex during precursor mRNA splicing and plays a role in mRNA surveillance in the cytoplasm. Using the Y14/Magoh heterodimer as bait in a screening for its interacting partners, we identified the protein-arginine methyltransferase PRMT5 as a candidate. We show that Y14 and Magoh, but not other factors of the exon junction complex, interact with the cytoplasmic PRMT5-containing methylosome. We further provide evidence that Y14 promoted the activity of PRMT5 in methylation of Sm proteins of the small nuclear ribonucleoprotein core, whereas knockdown of Y14 reduced their methylation level. Moreover, Y14 overexpression induced the formation of a large, active, and small nuclear ribonucleoprotein (snRNP)-associated methylosome complex. However, Y14 may only transiently associate with the snRNP assembly complex in the cytoplasm. Together, our results suggest that Y14 facilitates Sm protein methylation probably by its activity in promoting the formation or stability of the methylosome-containing complex. We hypothesize that Y14 provides a regulatory link between pre-mRNA splicing and snRNP biogenesis.

TRAP150 activates pre-mRNA splicing and promotes nuclear mRNA degradation.

TRAP150 has been identified as a subunit of the transcription regulatory complex TRAP/Mediator, and also a component of the spliceosome. The exact function of TRAP150, however, remains unclear. We recently identified TRAP150 by its association with the mRNA export factor TAP. TRAP150 contains an arginine/serine-rich domain and has sequence similarity with the cell death-promoting transcriptional repressor BCLAF1. We found that TRAP150 co-localizes with splicing factors in nuclear speckles, and is required for pre-mRNA splicing and activates splicing in vivo. TRAP150 remains associated with the spliced mRNA after splicing, and accordingly, it interacts with the integral exon junction complex. Unexpectedly, when tethered to a precursor mRNA, TRAP150 can trigger mRNA degradation in the nucleus. However, unlike nonsense-mediated decay, TRAP150-mediated mRNA decay is irrespective of the presence of upstream stop codons and occurs in the nucleus. Moreover, TRAP150 activates pre-mRNA splicing and induces mRNA degradation by its separable functional domains. Therefore, TRAP150 represents a multi-functional protein involved in nuclear mRNA metabolism.

DDX3 interacts with USP9X and participates in deubiquitination of the anti-apoptotic protein MCL1.

Here, we describe a novel interaction between the RNA helicase DDX3 and the deubiquitinase ubiquitin-specific peptidase 9 X-linked (USP9X) in human cells. Domain mapping studies reveal that the C-terminal region of DDX3 interacted with the N terminus of USP9X. USP9X was predominantly localized in the cytoplasm where the interaction between DDX3 and USP9X occurred. USP9X was not visibly enriched in cytoplasmic stress granules (SGs) under oxidative stress conditions, whereas overexpression of GFP-DDX3 induced SG formation and recruited USP9X to SGs in HeLa cells. Luciferase reporter assays showed that depletion of USP9X had no significant effect on DDX3-mediated translation. Given that DDX3 is not ubiquitinated upon ubiquitin overexpression, it is unlikely that DDX3 serves as a substrate of USP9X. Importantly, we found that ubiquitinated MCL1 was accumulated upon depletion of USP9X and/or DDX3 in MG132-treated cells, suggesting that USP9X and DDX3 play a role in regulating MCL1 protein stability and anti-apoptotic function. This study indicates that DDX3 exerts anti-apoptotic effects probably by coordinating with USP9X in promoting MCL1 deubiquitination.

Alternative splicing in Acad8 resulting a mitochondrial defect and progressive hepatic steatosis in mice.

Using a combination of N-ethyl-N-nitrosourea-mediated mutagenesis and metabolomics-guided screening, we identified mice with elevated blood levels of short-chain C4-acylcarnitine and increased urine isobutyryl-glycine. Genome-wide homozygosity screening, followed by fine mapping, located the disease gene to 15-25 Mb of mouse chromosome 9 where a candidate gene, Acad8, encoding mitochondrial isobutyryl-CoA dehydrogenase was located. Genomic DNA sequencing revealed a single-nucleotide mutation at -17 of the first intron of Acad8 in affected mice. cDNA sequencing revealed an intronic 28-bp insertion at the site of the mutation, which caused a frame shift with a premature stop codon. In vitro splicing assay confirmed that the mutation was sufficient to activate an upstream, aberrant 3' splice site. There was a reduction in the expression of Acad8 at both the mRNA and protein levels. The mutant mice grew normally but demonstrated cold intolerance at young age with a progressive hepatic steatosis. Homozygous mutant mice hepatocytes had abnormal mitochondria with crystalline inclusions, suggestive of mitochondriopathy. This mouse model of isobutyryl-CoA dehydrogenase deficiency could provide us a better understanding of the possible role of IBD deficiency in mitochondriopathy and fatty liver.

A role for transportin in deposition of TTP to cytoplasmic RNA granules and mRNA decay.

Importin-beta family members, which shuttle between the nucleus and the cytoplasm, are essential for nucleocytoplasmic transport of macromolecules. We attempted to explore whether importin-beta family proteins change their cellular localization in response to environmental change. In this report, we show that transportin (TRN) was minimally detected in cytoplasmic processing bodies (P-bodies) under normal cell conditions but largely translocated to stress granules (SGs) in stressed cells. Fluorescence recovery after photobleaching analysis indicated that TRN moves rapidly in and out of cytoplasmic granules. Depletion of TRN greatly enhanced P-body formation but did not affect the number or size of SGs, suggesting that TRN or its cargo(es) participates in cellular function of P-bodies. Accordingly, TRN associated with tristetraprolin (TTP) and its AU-rich element (ARE)-containing mRNA substrates. Depletion of TRN increased the number of P-bodies and stabilized ARE-containing mRNAs, as observed with knockdown of the 5'-3' exonuclease Xrn1. Moreover, depletion of TRN retained TTP in P-bodies and meanwhile reduced the fraction of mobile TTP to SGs. Therefore, our data together suggest that TRN plays a role in trafficking of TTP between the cytoplasmic granules and whereby modulates the stability of ARE-containing mRNAs.