Conserved RNA Binding Protein Research Articles

Discrete LIN28 binding sites in mature messenger RNA sequences reveals regulation of a network of splicing factors and downstream alternative splicing patterns

LIN28 is a conserved RNA binding protein important for pluripotency, reprogramming and oncogenesis. Studies thus far have focused primarily on the roles of LIN28 through its regulation of the microRNA (miRNA) let‐7. Here, our goal was to identify and characterize the network of mature messenger RNA targets of LIN28 in order to explain its let‐7 independent regulatory mechanisms. Using UV cross‐linking and immunoprecipitation of the LIN28 protein followed by high‐throughput sequencing (CLIP‐seq) we have identified direct LIN28 binding regions on target RNAs in human embryonic stem cells and somatic cells expressing exogenous LIN28. We found that in addition to hundreds of miRNAs, LIN28 directly binds to thousands of human mRNA genes, including LIN28 itself and dozens of splicing regulators. Furthermore, we confirm in a set of targets that this binding results in an increase in translation. Finally, splicing‐sensitive junction arrays demonstrated that LIN28 over‐expression causes widespread downstream alternative splicing differences. These findings show that LIN28 contributes to cellular homeostasis via direct mRNA interactions and downstream AS events, alterations of which may result in cancer and differentiation.

The active stem cell specific expression of sponge Musashi homolog EflMsiA suggests its involvement in maintaining the stem cell state

A hallmark of stem cells is the ability to sustainably generate stem cells themselves (self-renew) as well as differentiated cells. Although a full understanding of this ability will require clarifying underlying the primordial molecular and cellular mechanisms, how stem cells maintain their stem state and their population in the evolutionarily oldest extant multicellular organisms, sponges, is poorly understood. Here, we report the identification of the first stem cell-specific gene in demosponges, a homolog of Musashi (an evolutionarily conserved RNA binding protein that regulates the stem cell state in various organisms). EflMsiA, a Musashi paralog, is specifically expressed in stem cells (archeocytes) in the freshwater sponge Ephydatia fluviatilis. EflMsiA protein is localized predominantly in the nucleus, with a small fraction in the cytoplasm, in archeocytes. When archeocytes enter M-phase, EflMsiA protein diffuses into the cytoplasm, probably because of the breakdown of the nuclear membrane. In the present study, the existence of two types of M-phase archeocytes [(M)-archeocytes] was revealed by a precise analysis of the expression levels of EflMsiA mRNA and protein. In Type I (M)-archeocytes, presumably archeocytes undergoing self-renewal, the expression levels of EflMsiA mRNA and protein were high. In Type II (M)-archeocytes, presumably archeocytes committed to differentiate (committed archeocytes), the expression levels of EflMsiA mRNA and protein were about 60% and 30% lower than those in Type I (M)-archeocytes. From these results, archeocytes can be molecularly defined for the first time as EflMsiA-mRNA-expressing cells. Furthermore, these findings shed light on the mode of cell division of archeocytes and suggest that archeocytes divide symmetrically for both self-renewal and differentiation.

TDP-43 Regulates Drosophila Neuromuscular Junctions Growth by Modulating Futsch/MAP1B Levels and Synaptic Microtubules Organization

TDP-43 is an evolutionarily conserved RNA binding protein recently associated with the pathogenesis of different neurological diseases. At the moment, neither its physiological role in vivo nor the mechanisms that may lead to neurodegeneration are well known. Previously, we have shown that TDP-43 mutant flies presented locomotive alterations and structural defects at the neuromuscular junctions. We have now investigated the functional mechanism leading to these phenotypes by screening several factors known to be important for synaptic growth or bouton formation. As a result we found that alterations in the organization of synaptic microtubules correlate with reduced protein levels in the microtubule associated protein futsch/MAP1B. Moreover, we observed that TDP-43 physically interacts with futsch mRNA and that its RNA binding capacity is required to prevent futsch down regulation and synaptic defects.

La-related protein 4 binds poly(A), interacts with the poly(A)-binding protein MLLE domain via a variant PAM2w motif, and can promote mRNA stability.

The conserved RNA binding protein La recognizes UUU-3'OH on its small nuclear RNA ligands and stabilizes them against 3'-end-mediated decay. We report that newly described La-related protein 4 (LARP4) is a factor that can bind poly(A) RNA and interact with poly(A) binding protein (PABP). Yeast two-hybrid analysis and reciprocal immunoprecipitations (IPs) from HeLa cells revealed that LARP4 interacts with RACK1, a 40S ribosome- and mRNA-associated protein. LARP4 cosediments with 40S ribosome subunits and polyribosomes, and its knockdown decreases translation. Mutagenesis of the RNA binding or PABP interaction motifs decrease LARP4 association with polysomes. Several translation and mRNA metabolism-related proteins use a PAM2 sequence containing a critical invariant phenylalanine to make direct contact with the MLLE domain of PABP, and their competition for the MLLE is thought to regulate mRNA homeostasis. Unlike all ∼150 previously analyzed PAM2 sequences, LARP4 contains a variant PAM2 (PAM2w) with tryptophan in place of the phenylalanine. Binding and nuclear magnetic resonance (NMR) studies have shown that a peptide representing LARP4 PAM2w interacts with the MLLE of PABP within the affinity range measured for other PAM2 motif peptides. A cocrystal of PABC bound to LARP4 PAM2w shows tryptophan in the pocket in PABC-MLLE otherwise occupied by phenylalanine. We present evidence that LARP4 expression stimulates luciferase reporter activity by promoting mRNA stability, as shown by mRNA decay analysis of luciferase and cellular mRNAs. We propose that LARP4 activity is integrated with other PAM2 protein activities by PABP as part of mRNA homeostasis.

Regulation of lin-4 miRNA expression, organismal growth and development by a conserved RNA binding protein in C. elegans

Transcription and multiple processing steps are required to produce specific 22 nucleotide microRNAs (miRNAs) that can regulate the expression of target genes. In C. elegans, mature lin-4 miRNA accumulates at the end of the first larval stage to repress its direct targets lin-14 and lin-28, allowing the progression of several somatic cell types to later larval fates. In this study, we characterized the expression of endogenous lin-4 and found that temporally regulated independent transcripts, but not constitutive lin-4 containing RNAs derived from an overlapping gene, are processed to mature lin-4 miRNA. Through an RNAi screen, we identified a conserved RNA binding protein gene rbm-28 (R05H10.2), homologous to the human RBM28 and yeast Nop4p proteins, that is important for lin-4 expression in C. elegans. We also demonstrate that rbm-28 genetically interacts with the lin-4 developmental timing pathway and uncover a previously unrecognized role for lin-14 and lin-28 in coordinating organismal growth.

Structure/function analysis of TDP-43 neurotoxicity in C. elegans

TDP-43 is a conserved RNA binding protein with known roles in mRNA splicing and stability. Cytoplasmic deposition of TDP-43 has been linked to multiple neurodegenerative diseases, including ALS and frontotemporal lobar dementia (FTLD). We have engineered pan-neuronal expression of human TDP-43 protein in C. elegans, with the goal of generating a convenient in vivo model of TDP-43 neurotoxicity. Full-length (wild type) human TDP-43 expressed in C. elegans is nuclear as is observed in human cells. Transgenic worms with neuronal human TDP-43 expression exhibit an uncoordinated phenotype and have abnormal motorneuron synapses. By using this uncoordinated phenotype as a read-out of TDP-43 neurotoxicity, we have investigated the contribution of specific TDP-43 domains as well as TDP-43 sub-cellular localization to toxicity. Deletion of either RNA recognition domain (RRM1 or RRM2) completely blocks neurotoxicity, as does deletion of the C-terminal region. These deleted TDP-43 variants still accumulate in the nucleus, although their subnuclear distribution is altered. In contrast, N-terminal deletions result in the formation of toxic cytoplasmic aggregates. Mutation of the TDP-43 nuclear localization signal (NLS) results in cytoplasmic deposition of fulllength TDP-43, which is not toxic. Mutations that alter two TDP-43 caspase cleavage sites (D89/219E), however, do not reverse TDP43 toxicity. Our results demonstrate that TDP-43 neurotoxicity can result from either nuclear activity of the full-length protein or accumulation of cytoplasmic aggregates composed of C-terminal fragments. These results suggest that there may be (at least) two different mechanisms of TDP-43 neurotoxicity.

The evolutionarily conserved RNA binding protein SMOOTH is essential for maintaining normal muscle function

The Drosophila smooth gene encodes an RNA binding protein which has been well conserved through evolution. To investigate the pleiotropic functions mediated by the smooth gene, we have selected and characterized two sm mutants which are viable as adults yet display robust phenotypes (including a significant decrease in lifespan). Utilizing these mutants, we have made the novel observation that disruption of the smooth/CG9218 locus leads to age-dependent muscle degeneration, and motor dysfunction. Histological characterization of adult sm mutants revealed marked abnormalities in the major thoracic tubular muscle: the tergal depressor of the trochanter (TDT). Corresponding defects include extensive loss/disruption of striations and nuclei. These pathological changes are recapitulated in flies that express a smooth RNA interference construct (sm RNAi) in the mesoderm. In contrast, targeting sm RNAi constructs to motor neurons does not alter muscle morphology. In addition to examining the TDT phenotype, we explored whether other muscular abnormalities were evident. Utilizing physiological assays developed in the laboratory, we have found that the thoracic muscle defect is preceded by dysmotility of the gastrointestinal tract. SMOOTH thus joins a growing list of hnRNPs that have previously been linked to muscle physiology/ pathophysiology. Our findings in Drosophila set the stage for investigating the role of the corresponding mammalian homolog, hnRNP L, in muscle function.

Expression and functional analysis of musashi-like genes in planarian CNS regeneration

The remarkable regenerative ability of planarians is made possible by a system of pluripotent stem cells. Recent molecular biological and ultrastructural studies have revealed that planarian stem cells consist of heterogeneous populations, which can be classified into several subsets according to their differential expression of RNA binding protein genes. In this study, we focused on planarian musashi family genes. Musashi encodes an evolutionarily conserved RNA binding protein known to be expressed in neural lineage cells, including neural stem cells, in many animals. Here, we investigated whether planarian musashi-like genes can be used as markers for detecting neural fate-restricted cells. Three musashi family genes, DjmlgA, DjmlgB and DjmlgC ( Dugesia japonica musashi-like gene A, B, C), and Djdmlg ( Dugesia japonica DAZAP-like/ musashi-like gene) were obtained by searching a planarian EST database and 5′ RACE, and each was found to have two RNA recognition motifs. We analyzed the types of cells expressing DjmlgA, DjmlgB, DjmlgC and Djdmlg by in situ hybridization, RT-PCR and single-cell RT-PCR analysis. Although Djdmlg was expressed in X-ray-sensitive stem cells and various types of differentiated cells, expression of the other three musashi-like genes was restricted to neural cells, as we expected. Further detailed analyses yielded the unexpected finding that these three planarian musashi family genes were predominantly expressed in X-ray-resistant differentiated neurons, but not in X-ray-sensitive stem cells. RNAi experiments suggested that these planarian musashi family genes might be involved in neural cell differentiation after neural cell-fate commitment.

Assignment of 1H, 13C, and 15N resonances of the RNA binding protein Snu13p from Saccharomyces cerevisiae

Snu13p is a highly conserved RNA binding protein from Saccharomyces cerevisiae required for both eukaryotic pre-mRNA splicing and pre-rRNA processing. The 1H, 13C, and 15N assignments were determined from multidimensional, multinuclear NMR experiments conducted at 25 degrees C.

Analysis of the in vivo function of the zebrafish fragile X gene family

Fragile X Syndrome is the leading cause of inherited mental retardation in humans. This X-linked disorder results from underexpression of the Fragile X mental retardation related protein(FMR1P) due to expansion and hypermethylation of a CGG trinucleotide repeat in the 5V UTR region of the Fragile X gene(fmr1). Fmr1 has two autosomal homologs, fxr1 and fxr2, which together comprise a family of highly conserved RNAbinding proteins thought to be involved with mRNA transport and translation, although their precise function(s) in development have not been determined. Expression analyses of these three zebrafish genes have been conducted by this laboratory and others. We have begun an analysis of the in vivo role of the Fragile X gene family in early zebrafish development through the use of morpholino(mo) oligonucleotide knockdowns. We are injecting 1–8 cell stage zebrafish embryos with mo-derived anti-sense reagents designed against the translation start sites of the three zebrafish Fragile Xrelated genes. Injected embryos are assayed for gross morphologic defects, as well as changes in organization of the nervous system through detection of axon tracts with anti-acetylated tubulin antibody. The well-described development of the early zebrafish nervous system, along with the ability to study dose-dependent inactivation of these target sequences individually and in combination should be powerful tools to help discern the in vivo function(s) of the Fragile X gene family in development.

The Drosophila Fragile X Mental Retardation Protein Controls Actin Dynamics by Directly Regulating Profilin in the Brain

Loss of Fragile X mental retardation protein (FMRP) function causes the highly prevalent Fragile X syndrome [1 and 2]. Identifying targets for the RNA binding FMRP is a major challenge and an important goal of research into the pathology of the disease. Perturbations in neuronal development and circadian behavior are seen in Drosophila dfmr1 mutants. Here we show that regulation of the actin cytoskeleton is under dFMRP control. dFMRP binds the mRNA of the Drosophila profilin homolog and negatively regulates Profilin protein expression. An increase in Profilin mimics the phenotype of dfmr1 mutants. Conversely, decreasing Profilin levels suppresses dfmr1 phenotypes. These data place a new emphasis on actin misregulation as a major problem in fmr1 mutant neurons.

Tab2 is a novel conserved RNA binding protein required for translation of the chloroplast psaB mRNA.

The chloroplast psaB mRNA encodes one of the reaction centre polypeptides of photosystem I. Protein pulse-labelling profiles indicate that the mutant strain of Chlamydomonas reinhardtii, F14, affected at the nuclear locus TAB2, is deficient in the translation of psaB mRNA and thus deficient in photosystem I activity. Genetic studies reveal that the target site for Tab2 is situated within the psaB 5'UTR. We have used genomic complementation to isolate the nuclear Tab2 gene. The deduced amino acid sequence of Tab2 (358 residues) displays 31-46% sequence identity with several orthologues found only in eukaryotic and prokaryotic organisms performing oxygenic photosynthesis. Directed mutagenesis indicates the importance of a highly conserved C-terminal tripeptide in Tab2 for normal psaB translation. The Tab2 protein is localized in the chloroplast stroma where it is associated with a high molecular mass protein complex containing the psaB mRNA. Gel mobility shift assays reveal a direct and specific interaction between Tab2 and the psaB 5'UTR. We propose that Tab2 plays a key role in the initial steps of PsaB translation and photosystem I assembly.

Legionella pneumophila CsrA is a pivotal repressor of transmission traits and activator of replication.

Legionella pneumophila can replicate inside amoebae and also alveolar macrophages to cause Legionnaires' Disease in susceptible hosts. When nutrients become limiting, a stringent-like response coordinates the differentiation of L. pneumophila to a transmissive form, a process mediated by the two-component system LetA/S and the sigma factors RpoS and FliA. Here we demonstrate that the broadly conserved RNA binding protein CsrA is a global repressor of L. pneumophila transmission phenotypes and an essential activator of intracellular replication. By analysing csrA expression and the phenotypes of csrA single and double mutants and a strain that expresses csrA constitutively, we demonstrate that, during replication in broth, CsrA represses every post-exponential phase phenotype examined, including cell shape shortening, motility, pigmentation, stress resistance, sodium sensitivity, cytotoxicity and efficient macrophage infection. At the transition to the post-exponential phase, LetA/S relieves CsrA repression to induce transmission phenotypes by both FliA-dependent and -independent pathways. For L. pneumophila to avoid lysosomal degradation in macrophages, CsrA repression must be relieved by LetA/S before phagocytosis; conversely, before intracellular bacteria can replicate, CsrA repression must be restored. The reciprocal regulation of replication and transmission exemplified by CsrA likely enhances the fitness of microbes faced with fluctuating environments.

Expression of the highly conserved RNA binding protein koc in embryogenesis

Expression of the highly conserved RNA binding protein koc in embryogenesis

Expression of the highly conserved RNA binding protein KOC in embryogenesis

The human KOC gene which is highly expressed in cancer shows typical structural features of an RNA binding protein. We analyzed the temporal and spatial expression pattern of KOC in mouse embryos at different gestational ages. The expression of KOC seems to be ubiquitous at early stages. During advanced gestation highest KOC expression occurs in the gut, pancreas, kidney, and in the developing brain. The expression pattern of KOC was compared to its Xenopus homologue Vg1-RBP during frog development. Similar expression was found in these organs suggesting an important functional role of the homologous proteins in embryonic development.

Lumleian Lectures on the Natural History and Diagnosis of Intrathoracic Cancer

<h3>Abstract</h3> Memory and learning involve activity-driven expression of proteins and cytoskeletal reorganisation at new synapses, often requiring post-transcriptional regulation a long distance from corresponding nuclei. A key factor expressed early in synapse formation is Msp300/Nesprin-1, which organises actin filaments around the new synapse. How Msp300 expression is regulated during synaptic plasticity is not yet known. Here, we show that the local translation of <i>msp300</i> is promoted during activity-dependent plasticity by the conserved RNA binding protein Syncrip/hnRNP Q, which binds to <i>msp300</i> transcripts and is essential for plasticity. Single molecule imaging shows that Syncrip is associated <i>in vivo</i> with <i>msp300</i> mRNA in ribosome-rich particles. Elevated neural activity alters the dynamics of Syncrip RNP granules at the synapse, suggesting a change in particle composition or binding that facilitates translation. These results introduce Syncrip as an important early-acting activity-dependent translational regulator of a plasticity gene that is strongly associated with human ataxias. <h3>Syncrip regulates synaptic plasticity via <i>msp300</i></h3> Titlow et al. find that Syncrip (hnRNPQ RNA binding protein) acts directly on <i>msp300</i> to modulate activity-dependent synaptic plasticity. <i>In vivo</i> biophysical experiments reveal activity-dependent changes in RNP complex sizes compatible with an increase in translation at the synapse.