Person:

Reed, Robin

The conserved TREX complex, which contains UAP56, Aly, CIP29, and the multi-subunit THO complex, functions in mRNA export. Recently, several putative new components of the human TREX complex were identified by mass spectrometry. Here, we investigated the function of two of these, PDIP3 and ZC11A. Our data indicate that both of these proteins are components of a common TREX complex and function in mRNA export. Recently, we found that both CIP29 and Aly associate with the DEAD box helicase UAP56 and with the TREX complex in an ATP-dependent manner. We now show that this is also the case for PDIP3 and ZC11A. Thus, together with previous work, our data indicate that the TREX complex participates in multiple ATP-dependent interactions.

ALS is a devastating neurodegenerative disease whose causes are still poorly understood. To identify additional genetic risk factors, here we assess the role of de novo mutations in ALS by sequencing the exomes of 47 ALS patients and both of their unaffected parents (n=141 exomes). We found that amino acid-altering de novo mutations are enriched in genes encoding chromatin regulators, including the neuronal chromatin remodeling complex component SS18L1/CREST. CREST mutations inhibit activity-dependent neurite outgrowth in primary neurons, and CREST associates with the ALS protein FUS. These findings expand our understanding of the ALS genetic landscape and provide a resource for future studies into the pathogenic mechanisms contributing to sporadic ALS.

The metazoan TREX complex is recruited to mRNA during nuclear RNA processing and functions in exporting mRNA to the cytoplasm. Nxf1 is an mRNA export receptor, which binds processed mRNA and transports it through the nuclear pore complex. At present, the relationship between TREX and Nxf1 is not understood. Here we show that Nxf1 uses an intramolecular interaction to inhibit its own RNA binding activity. When the TREX subunits Aly and Thoc5 make contact with Nxf1, Nxf1 is driven into an open conformation, exposing its RNA binding domain, allowing RNA binding. Moreover, the combined knockdown of Aly and Thoc5 drastically reduces the amount of Nxf1 bound to mRNA in vivo and also causes a severe mRNA export block. Together, our data indicate that TREX provides a license for mRNA export by driving Nxf1 into a conformation capable of binding mRNA.

We previously showed that mRNAs synthesized from three genes that naturally lack introns contain a portion of their coding sequence, known as a cytoplasmic accumulation region (CAR), which is essential for stable accumulation of the intronless mRNAs in the cytoplasm. The CAR in each mRNA is unexpectedly large, ranging in size from ∼160 to 285 nt. Here, we identified one or more copies of a 10-nt consensus sequence in each CAR. To determine whether this element (designated CAR-E) functions in cytoplasmic accumulation of intronless mRNA, we multimerized the most conserved CAR-E and inserted it upstream of β-globin cDNA, which is normally retained/degraded in the nucleus. Significantly, the tandem CAR-E, but not its antisense counterpart, rescued cytoplasmic accumulation of β-globin cDNA transcripts. Moreover, dinucleotide mutations in the CAR-E abolished this rescue. We show that the CAR-E, but not the mutant CAR-E, associates with components of the TREX mRNA export machinery, the Prp19 complex and U2AF2. Moreover, knockdown of these factors results in nuclear retention of the intronless mRNAs. Together, these data suggest that the CAR-E promotes export of intronless mRNA by sequence-dependent recruitment of the mRNA export machinery.

Previous studies in vivo reported that processing of primary microRNA (pri-miRNA) is coupled to transcription by RNA polymerase II (RNAP II) and can occur co-transcriptionally. Here we have established a robust in vivo system in which pri-miRNA is transcribed by RNAP II and processed to pre-miRNA in HeLa cell nuclear extracts. We show that both the kinetics and efficiency of pri-miRNA processing are dramatically enhanced in this system compared to that of the corresponding naked pri-miRNA. Moreover, this enhancement is general as it occurs with multiple pri-miRNAs. We also show that nascent pri-miRNA is efficiently processed before it is released from the DNA template. Together, our work directly demonstrates that transcription and pri-miRNA processing are functionally coupled and establishes the first in vivo model systems for this functional coupling and for co-transcriptional processing.

Mutations in FUS cause amyotrophic lateral sclerosis (ALS), but the molecular pathways leading to neurodegeneration remain obscure. We previously found that U1 snRNP is the most abundant FUS interactor. Here, we report that components of the U1 snRNP core particle (Sm proteins and U1 snRNA), but not the mature U1 snRNP-specific proteins (U1-70K, U1A and U1C), co-mislocalize with FUS to the cytoplasm in ALS patient fibroblasts harboring mutations in the FUS nuclear localization signal (NLS). Similar results were obtained in HeLa cells expressing the ALS-causing FUS R495X NLS mutation, and mislocalization of Sm proteins is RRM-dependent. Moreover, as observed with FUS, knockdown of any of the U1 snRNP-specific proteins results in a dramatic loss of SMN-containing Gems. Significantly, knockdown of U1 snRNP in zebrafish results in motor axon truncations, a phenotype also observed with FUS, SMN and TDP-43 knockdowns. Our observations linking U1 snRNP to ALS patient cells with FUS mutations, SMN-containing Gems, and motor neurons indicate that U1 snRNP is a component of a molecular pathway associated with motor neuron disease. Linking an essential canonical splicing factor (U1 snRNP) to this pathway provides strong new evidence that splicing defects may be involved in pathogenesis and that this pathway is a potential therapeutic target.

Genomic instability is a hallmark of human cancer, and results in widespread somatic copy number alterations. We used a genome-scale shRNA viability screen in human cancer cell lines to systematically identify genes that are essential in the context of particular copy-number alterations (copy-number associated gene dependencies). The most enriched class of copy-number associated gene dependencies was CYCLOPS (Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS) genes, and spliceosome components were the most prevalent. One of these, the pre-mRNA splicing factor SF3B1, is also frequently mutated in cancer. We validated SF3B1 as a CYCLOPS gene and found that human cancer cells harboring partial SF3B1 copy-loss lack a reservoir of SF3b complex that protects cells with normal SF3B1 copy number from cell death upon partial SF3B1 suppression. These data provide a catalog of copy-number associated gene dependencies and identify partial copy-loss of wild-type SF3B1 as a novel, non-driver cancer gene dependency. DOI: http://dx.doi.org/10.7554/eLife.23268.001

The conserved RNA helicase DDX3 is of major medical importance due to its involvement in numerous cancers, human hepatitis C virus (HCV) and HIV. Although DDX3 has been reported to have a wide variety of cellular functions, its precise role remains obscure. Here, we raised a new antibody to DDX3 and used it to show that DDX3 is evenly distributed throughout the cytoplasm at steady state. Consistent with this observation, HA-tagged DDX3 also localizes to the cytoplasm. RNAi of DDX3 in both human and Drosophila cells shows that DDX3 is required for cell viability. Moreover, using RNAi, we show that DDX3 is required for expression of protein from reporter constructs. In contrast, we did not detect a role for DDX3 in nuclear steps in gene expression. Further insight into the function of DDX3 came from the observation that its major interaction partner is the multi-component translation initiation factor eIF3. We conclude that a primary function for DDX3 is in protein translation, via an interaction with eIF3.

Mutations in multiple RNA/DNA binding proteins cause Amyotrophic Lateral Sclerosis (ALS). Included among these are the three members of the FET family (FUS, EWSR1 and TAF15) and the structurally similar MATR3. Here, we characterized the interactomes of these four proteins, revealing that they largely have unique interactors, but share in common an association with U1 snRNP. The latter observation led us to analyze the interactome of the U1 snRNP machinery. Surprisingly, this analysis revealed the interactome contains ~220 components, and of these, >200 are shared with the RNA polymerase II (RNAP II) machinery. Among the shared components are multiple ALS and Spinal muscular Atrophy (SMA)-causative proteins and numerous discrete complexes, including the SMN complex, transcription factor complexes, and RNA processing complexes. Together, our data indicate that the RNAP II/U1 snRNP machinery functions in a wide variety of molecular pathways, and these pathways are candidates for playing roles in ALS/SMA pathogenesis.