Cancer-associated snaR-A noncoding RNA interacts with core splicing machinery and disrupts processing of mRNA subpopulations.

RNA polymerase III (Pol III) activity in cancer is linked to the production of small noncoding (nc)RNAs that are otherwise silent in most tissues. snaR-A (small NF90-associated RNA isoform A) - a hominid-specific ncRNA shown to enhance cell proliferation, migration, and invasion - is a cancer-emergent Pol III product that remains largely uncharacterized despite promoting growth phenotypes. Here, we applied a combination of genomic and biochemical approaches to study the biogenesis and subsequent protein interactions of snaR-A and to better understand its role as a putative driver of cancer progression. By profiling the chromatin landscapes across a multitude of primary tumor types, we show that predicted snaR-A upregulation is broadly linked with unfavorable outcomes among cancer patients. At the molecular level, we unexpectedly discover widespread interactions between snaR-A and mRNA splicing factors, including SF3B2 - a core component of the U2 small nuclear ribonucleoprotein (snRNP). We find that SF3B2 levels are sensitive to high snaR-A abundance and that depletion of snaR-A alone is sufficient to decrease intron retention levels across subpopulations of mRNA enriched for U2 snRNP occupancy. snaR-A sensitive genes are characterized by high GC content, close spatial proximity to nuclear bodies concentrated in pre-mRNA splicing factors, and functional enrichment for proteins involved in deacetylation and autophagy. We highlight examples of splicing misregulation and increased protein levels following snaR-A depletion for a wide-ranging set of factors, suggesting snaR-A-driven splicing defects may have far-reaching effects that re-shape the cellular proteome. These findings clarify the molecular activities and consequences of snaR-A in cancer, and altogether establish a novel mechanism through which Pol III overactivity may promote tumorigenesis.

Cytoplasmic Relocalization and Colocalization with Viroplasms of Host Cell Proteins, and Their Role in Rotavirus Infection.

Rotavirus replicates in the cytoplasm of infected cells in unique virus-induced cytoplasmic inclusion bodies called viroplasms (VMs), which are nucleated by two essential viral nonstructural proteins, NSP2 and NSP5. However, the precise composition of the VM, the intracellular localization of host proteins during virus infection, and their association with VMs or role in rotavirus growth remained largely unexplored. Mass spectrometry analyses revealed the presence of several host heterogeneous nuclear ribonucleoproteins (hnRNPs), AU-rich element-binding proteins (ARE-BPs), and cytoplasmic proteins from uninfected MA104 cell extracts in the pulldown (PD) complexes of the purified viroplasmic proteins NSP2 and NSP5. Immunoblot analyses of PD complexes from RNase-treated and untreated cell extracts, analyses of coimmunoprecipitation complexes using RNase-treated infected cell lysates, and direct binding assays using purified recombinant proteins further demonstrated that the interactions of the majority of the hnRNPs and ARE-BPs with viroplasmic proteins are RNA independent. Time course immunoblot analysis of the nuclear and cytoplasmic fractions from rotavirus-infected and mock-infected cells and immunofluorescence confocal microscopy analyses of virus-infected cells revealed a surprising sequestration of the majority of the relocalized host proteins in viroplasms. Analyses of ectopic overexpression and small interfering RNA (siRNA)-mediated downregulation of expression revealed that host proteins either promote or inhibit viral protein expression and progeny virus production in virus-infected cells. This study demonstrates that rotavirus induces the cytoplasmic relocalization and sequestration of a large number of nuclear and cytoplasmic proteins in viroplasms, subverting essential cellular processes in both compartments to promote rapid virus growth, and reveals that the composition of rotavirus viroplasms is much more complex than is currently understood.IMPORTANCE Rotavirus replicates exclusively in the cytoplasm. Knowledge on the relocalization of nuclear proteins to the cytoplasm or the role(s) of host proteins in rotavirus infection is very limited. In this study, it is demonstrated that rotavirus infection induces the cytoplasmic relocalization of a large number of nuclear RNA-binding proteins (hnRNPs and AU-rich element-binding proteins). Except for a few, most nuclear hnRNPs and ARE-BPs, nuclear transport proteins, and some cytoplasmic proteins directly interact with the viroplasmic proteins NSP2 and NSP5 in an RNA-independent manner and become sequestered in the viroplasms of infected cells. The host proteins differentially affected viral gene expression and virus growth. This study demonstrates that rotavirus induces the relocalization and sequestration of a large number of host proteins in viroplasms, affecting host processes in both compartments and generating conditions conducive for virus growth in the cytoplasm of infected cells.

Maternal Dead-end 1 promotes translation of nanos1 by binding the eIF3 complex.

In the developing embryo, primordial germ cells (PGCs) represent the exclusive progenitors of the gametes, and their loss results in adult infertility. During early development, PGCs are exposed to numerous signals that specify somatic cell fates. To prevent somatic differentiation, PGCs must transiently silence their genome, an early developmental process that requires Nanos activity. However, it is unclear how Nanos translation is regulated in developing embryos. We report here that translation of nanos1 after fertilization requires Dead-end 1 (Dnd1), a vertebrate-specific germline RNA-binding protein. We provide evidence that Dnd1 protein, expression of which is low in oocytes, but increases dramatically after fertilization, directly interacts with, and relieves the inhibitory function of eukaryotic initiation factor 3f, a repressive component in the 43S preinitiation complex. This work uncovers a novel translational regulatory mechanism that is fundamentally important for germline development.

Poly(A) tail length regulates PABPC1 expression to tune translation in the heart.

The rate of protein synthesis in the adult heart is one of the lowest in mammalian tissues, but it increases substantially in response to stress and hypertrophic stimuli through largely obscure mechanisms. Here, we demonstrate that regulated expression of cytosolic poly(A)-binding protein 1 (PABPC1) modulates protein synthetic capacity of the mammalian heart. We uncover a poly(A) tail-based regulatory mechanism that dynamically controls PABPC1 protein synthesis in cardiomyocytes and thereby titrates cellular translation in response to developmental and hypertrophic cues. Our findings identify PABPC1 as a direct regulator of cardiac hypertrophy and define a new paradigm of gene regulation in the heart, where controlled changes in poly(A) tail length influence mRNA translation.

ESRP2 controls an adult splicing programme in hepatocytes to support postnatal liver maturation.

Although major genetic networks controlling early liver specification and morphogenesis are known, the mechanisms responsible for postnatal hepatic maturation are poorly understood. Here we employ global analyses of the mouse liver transcriptome to demonstrate that postnatal remodelling of the liver is accompanied by large-scale transcriptional and post-transcriptional transitions that are cell-type-specific and temporally coordinated. Combining detailed expression analyses with gain- and loss-of-function studies, we identify epithelial splicing regulatory protein 2 (ESRP2) as a conserved regulatory factor that controls the neonatal-to-adult switch of ∼20% of splice isoforms in mouse and human hepatocytes. The normal shift in splicing coincides tightly with dramatic postnatal induction of ESRP2 in hepatocytes. We further demonstrate that forced expression of ESRP2 in immature mouse and human hepatocytes is sufficient to drive a reciprocal shift in splicing and causes various physiological abnormalities. These findings define a direct role for ESRP2 in the generation of conserved repertoires of adult splice isoforms that facilitate terminal differentiation and maturation of hepatocytes.