Mud2 functions in transcription by recruiting the Prp19 and TREX complexes to transcribed genes

The different steps of gene expression are intimately linked to coordinate and regulate this complex process. During transcription, numerous RNA-binding proteins are already loaded onto the nascent mRNA and package the mRNA into a messenger ribonucleoprotein particle (mRNP). These RNA-binding proteins are often also involved in other steps of gene expression than mRNA packaging. For example, TREX functions in transcription, mRNP packaging and nuclear mRNA export. Previously, we showed that the Prp19 splicing complex (Prp19C) is needed for efficient transcription as well as TREX occupancy at transcribed genes. Here, we show that the splicing factor Mud2 interacts with Prp19C and is needed for Prp19C occupancy at transcribed genes in Saccharomyces cerevisiae. Interestingly, Mud2 is not only recruited to intron-containing but also to intronless genes indicating a role in transcription. Indeed, we show for the first time that Mud2 functions in transcription. Furthermore, these functions of Mud2 are likely evolutionarily conserved as Mud2 is also recruited to an intronless gene and interacts with Prp19C in Drosophila melanogaster. Taken together, we classify Mud2 as a novel transcription factor that is necessary for the recruitment of mRNA-binding proteins to the transcription machinery. Thus, Mud2 is a multifunctional protein important for transcription, splicing and most likely also mRNP packaging

Promotion of importin α–mediated nuclear import by the phosphorylation-dependent binding of cargo protein to 14-3-3

14-3-3 proteins are phosphoserine/threonine-binding proteins that play important roles in many regulatory processes, including intracellular protein targeting. 14-3-3 proteins can anchor target proteins in the cytoplasm and in the nucleus or can mediate their nuclear export. So far, no role for 14-3-3 in mediating nuclear import has been described. There is also mounting evidence that nuclear import is regulated by the phosphorylation of cargo proteins, but the underlying mechanism remains elusive. Myopodin is a dual-compartment, actin-bundling protein that functions as a tumor suppressor in human bladder cancer. In muscle cells, myopodin redistributes between the nucleus and the cytoplasm in a differentiation-dependent and stress-induced fashion. We show that importin α binding and the subsequent nuclear import of myopodin are regulated by the serine/threonine phosphorylation-dependent binding of myopodin to 14-3-3. These results establish a novel paradigm for the promotion of nuclear import by 14-3-3 binding. They provide a molecular explanation for the phosphorylation-dependent nuclear import of nuclear localization signal-containing cargo proteins

Extraction of protein profiles from primary neurons using active contour models and wavelets

AbstractThe function of complex networks in the nervous system relies on the proper formation of neuronal contacts and their remodeling. To decipher the molecular mechanisms underlying these processes, it is essential to establish unbiased automated tools allowing the correlation of neurite morphology and the subcellular distribution of molecules by quantitative means.We developed NeuronAnalyzer2D, a plugin for ImageJ, which allows the extraction of neuronal cell morphologies from two dimensional high resolution images, and in particular their correlation with protein profiles determined by indirect immunostaining of primary neurons. The prominent feature of our approach is the ability to extract subcellular distributions of distinct biomolecules along neurites. To extract the complete areas of neurons, required for this analysis, we employ active contours with a new distance based energy. For locating the structural parts of neurons and various morphological parameters we adopt a wavelet based approach. The presented approach is able to extract distinctive profiles of several proteins and reports detailed morphology measurements on neurites.We compare the detected neurons from NeuronAnalyzer2D with those obtained by NeuriteTracer and Vaa3D-Neuron, two popular tools for automatic neurite tracing. The distinctive profiles extracted for several proteins, for example, of the mRNA binding protein ZBP1, and a comparative evaluation of the neuron segmentation results proves the high quality of the quantitative data and proves its practical utility for biomedical analyses

Activation of oligodendroglial Fyn kinase enhances translation of mRNAs transported in hnRNP A2–dependent RNA granules

Central nervous system myelination requires the synthesis of large amounts of myelin basic protein (MBP) at the axon–glia contact site. MBP messenger RNA (mRNA) is transported in RNA granules to oligodendroglial processes in a translationally silenced state. This process is regulated by the trans-acting factor heterogeneous nuclear ribonucleoprotein (hnRNP) A2 binding to the cis-acting A2 response element (A2RE). Release of this repression of MBP mRNA translation is thus essential for myelination. Mice deficient in the Src family tyrosine kinase Fyn are hypomyelinated and contain reduced levels of MBP. Here, we identify hnRNP A2 as a target of activated Fyn in oligodendrocytes. We show that active Fyn phosphorylates hnRNP A2 and stimulates translation of an MBP A2RE–containing reporter construct. Neuronal adhesion molecule L1 binding to oligodendrocytes results in Fyn activation, which leads to an increase in hnRNP A2 phosphorylation. These results suggest that Fyn kinase activation results in the localized translation of MBP mRNA at sites of axon–glia contact and myelin deposition

Characterization of the actin binding properties of the vasodilator-stimulated phosphoprotein VASP

AbstractThe vasodilator-stimulated phosphoprotein (VASP) colocalizes with the ends of stress fibers in cell-matrix and cell-cell contacts. We report here that bacterially expressed murine VASP directly interacts with skeletal muscle actin in several test systems including cosedimentation, viscometry and polymerization assays. It nucleates actin polymerization and tightly bundles actin filaments. The interaction with actin is salt-sensitive, indicating that the complex formation is primarily based on electrostatic interactions. Actin binding is confined to the C-terminal domain of VASP (EVH2). This domain, when expressed as a fusion protein with EGFP, associates with stress fibers in transiently transfected cells

Two ZBP1 KH domains facilitate β-actin mRNA localization, granule formation, and cytoskeletal attachment

Chicken embryo fibroblasts (CEFs) localize β-actin mRNA to their lamellae, a process important for the maintenance of cell polarity and motility. The localization of β-actin mRNA requires a cis localization element (zipcode) and involves zipcode binding protein 1 (ZBP1), a protein that specifically binds to the zipcode. Both localize to the lamellipodia of polarized CEFs. ZBP1 and its homologues contain two NH2-terminal RNA recognition motifs (RRMs) and four COOH-terminal hnRNP K homology (KH) domains. By using ZBP1 truncations fused to GFP in conjunction with in situ hybridization analysis, we have determined that KH domains three and four were responsible for granule formation and cytoskeletal association. When the NH2 terminus was deleted, granules formed by the KH domains alone did not accumulate at the leading edge, suggesting a role for the NH2 terminus in targeting transport granules to their destination. RNA binding studies were used to show that the third and fourth KH domains, not the RRM domains, bind the zipcode of β-actin mRNA. Overexpression of the four KH domains or certain subsets of these domains delocalized β-actin mRNA in CEFs and inhibited fibroblast motility, demonstrating the importance of ZBP1 function in both β-actin mRNA localization and cell motility

Raver1, a dual compartment protein, is a ligand for PTB/hnRNPI and microfilament attachment proteins

By screening a yeast two-hybrid library with COOH-terminal fragments of vinculin/metavinculin as the bait, we identified a new protein termed raver1. Raver1 is an 80-kD multidomain protein and widely expressed but to varying amounts in different cell lines. In situ and in vitro, raver1 forms complexes with the microfilament-associated proteins vinculin, metavinculin, and α-actinin and colocalizes with vinculin/metavinculin and α-actinin at microfilament attachment sites, such as cell–cell and cell matrix contacts of epithelial cells and fibroblasts, respectively, and in costameres of skeletal muscle. The NH2-terminal part of raver1 contains three RNA recognition motifs with homology to members of the heterogeneous nuclear RNP (hnRNP) family. Raver1 colocalizes with polypyrimidine tract binding protein (PTB)/hnRNPI, a protein involved in RNA splicing of microfilament proteins, in the perinucleolar compartment and forms complexes with PTB/hnRNPI. Hence, raver1 is a dual compartment protein, which is consistent with the presence of nuclear location signal and nuclear export sequence motifs in its sequence. During muscle differentiation, raver1 migrates from the nucleus to the costamere. We propose that raver1 may coordinate RNA processing and targeting as required for microfilament anchoring in specific adhesion sites

Maturation of mammalian H/ACA box snoRNAs: PAPD5-dependent adenylation and PARN-dependent trimming

Small nucleolar and small Cajal body RNAs (snoRNAs and scaRNAs) of the H/ACA box and C/D box type are generated by exonucleolytic shortening of longer precursors. Removal of the last few nucleotides at the 3' end is known to be a distinct step. We report that, in human cells, knock-down of the poly(A) specific ribonuclease (PARN), previously implicated only in mRNA metabolism, causes the accumulation of oligoadenylated processing intermediates of H/ACA box but not C/D box RNAs. In agreement with a role of PARN in snoRNA and scaRNA processing, the enzyme is concentrated in nucleoli and Cajal bodies. Oligo(A) tails are attached to a short stub of intron sequence remaining beyond the mature 3' end of the snoRNAs. The noncanonical poly(A) polymerase PAPD5 is responsible for addition of the oligo(A) tails. We suggest that deadenylation is coupled to clean 3' end trimming, which might serve to enhance snoRNA stability

The oncofetal RNA-binding protein IGF2BP1 is a druggable, post-transcriptional super-enhancer of E2F-driven gene expression in cancer

The IGF2 mRNA-binding protein 1 (IGF2BP1) is a non-catalytic post-transcriptional enhancer of tumor growth upregulated and associated with adverse prognosis in solid cancers. However, conserved effector pathway(s) and the feasibility of targeting IGF2BP1 in cancer remained elusive. We reveal that IGF2BP1 is a post-transcriptional enhancer of the E2F-driven hallmark in solid cancers. IGF2BP1 promotes G1/S cell cycle transition by stabilizing mRNAs encoding positive regulators of this checkpoint like E2F1. This IGF2BP1-driven shortening of the G1 cell cycle phase relies on 3′UTR-, miRNA- and m6A-dependent regulation and suggests enhancement of cell cycle progression by m6A-modifications across cancers. In addition to E2F transcription factors, IGF2BP1 also stabilizes E2F-driven transcripts directly indicating post-transcriptional 'super'-enhancer role of the protein in E2F-driven gene expression in cancer. The small molecule BTYNB disrupts this enhancer function by impairing IGF2BP1-RNA association. Consistently, BTYNB interferes with E2F-driven gene expression and tumor growth in experimental mouse tumor models

RNA Sequencing of Collecting Duct Renal Cell Carcinoma Suggests an Interaction between miRNA and Target Genes and a Predominance of Deregulated Solute Carrier Genes

Collecting duct carcinoma (CDC) is a rare renal cell carcinoma subtype with a very poor prognosis. There have been only a few studies on gene expression analysis in CDCs. We compared the gene expression profiles of two CDC cases with those of eight normal tissues of renal cell carcinoma patients. At a threshold of |log2fold-change| ≥1, 3349 genes were upregulated and 1947 genes were downregulated in CDCs compared to the normal samples. Pathway analysis of the deregulated genes revealed that cancer pathways and cell cycle pathways were most prominent in CDCs. The most upregulated gene was keratin 17, and the most downregulated gene was cubilin. Among the most downregulated genes were four solute carrier genes (SLC3A1, SLC9A3, SLC26A7, and SLC47A1). The strongest negative correlations between miRNAs and mRNAs were found between the downregulated miR-374b-5p and its upregulated target genes HIST1H3B, HK2, and SLC7A11 and between upregulated miR-26b-5p and its downregulated target genes PPARGC1A, ALDH6A1, and MARC2. An upregulation of HK2 and a downregulation of PPARGC1A, ALDH6A1, and MARC2 were observed at the protein level. Survival analysis of the cancer genome atlas (TCGA) dataset showed for the first time that low gene expression of MARC2, cubilin, and SLC47A1 and high gene expression of KRT17 are associated with poor overall survival in clear cell renal cell carcinoma patients. Altogether, we identified dysregulated protein-coding genes, potential miRNA-target interactions, and prognostic markers that could be associated with CDC