This service exclusively searches for literature that cites resources. Please be aware that the total number of searchable documents is limited to those containing RRIDs and does not include all open-access literature.
Splicing of the precursors of eukaryotic mRNA and some non-coding RNAs is catalyzed by the 'spliceosome', which comprises five RNA-protein complexes (small nuclear ribonucleoproteins, or snRNPs) that assemble in an ordered manner onto precursor-mRNAs. Much progress has been made in determining the gross morphology of spliceosomal assembly intermediates. Recently, the first crystal structure of a spliceosomal snRNP has provided significant insight into assembly and architecture of spliceosomal snRNPs in general and the structure-function relationship of human U1 snRNP in particular.
Coordinated assembly of viral and host factors is essential for the successful propagation of viruses as well as the generation of host antiviral response. Previous studies from our group, as well as from other groups, have identified host proteins interacting with various components of the hepatitis E virus (HEV). However, the functional relevance of host protein interactions in HEV replication context has been notably overlooked. The present study reports that heterogeneous nuclear ribonucleoproteins (hnRNPs), namely hnRNPK, hnRNPA2B1, hnRNPH, PCBP1 and PCBP2, interact with HEV RNA promoter and RNA-dependent RNA polymerase to regulate HEV replication. We found that hnRNPK and hnRNPA2B1 are the virus-supportive factors interacting with HEV RNA at promoter regions along with HEV polymerase protein, which are essential for HEV replication in the cells. Contrarily, hnRNPH, PCBP1 and PCBP2 are the antiviral factors that interact exclusively with HEV genomic promoter and inhibit HEV replication in Huh7 S10-3 cells. In vitro RNA-binding assays revealed that the antiviral hnRNP proteins hamper the binding of virus-supportive hnRNP proteins at HEV genomic promoter. In the binding reaction, the binding of HEV polymerase protein to the genomic promoter is slightly affected by the presence of antiviral hnRNPH. In an effort of visualizing the subcellular localization of hnRNP proteins in the HEV replication scenario in the Huh7 cells, we showed that hnRNPK, hnRNPA2B1, hnRNPH, PCBP1 and PCBP2 redistribute from nucleus to cytoplasm. In conclusion, our study highlights the importance of hnRNP proteins in HEV replication regulation.
The biological functions of poly(ADP-ribosyl)ation of heterogeneous nuclear ribonucleoproteins (hnRNPs) are not well understood. However, it is known that hnRNPs are involved in the regulation of alternative splicing for many genes, including the Ddc gene in Drosophila. Therefore, we first confirmed that poly(ADP-ribose) (pADPr) interacts with two Drosophila hnRNPs, Squid/hrp40 and Hrb98DE/hrp38, and that this function is regulated by Poly(ADP-ribose) Polymerase 1 (PARP1) and Poly(ADP-ribose) Glycohydrolase (PARG) in vivo. These findings then provided a basis for analyzing the role of pADPr binding to these two hnRNPs in terms of alternative splicing regulation. Our results showed that Parg null mutation does cause poly(ADP-ribosyl)ation of Squid and hrp38 protein, as well as their dissociation from active chromatin. Our data also indicated that pADPr binding to hnRNPs inhibits the RNA-binding ability of hnRNPs. Following that, we demonstrated that poly(ADP-ribosyl)ation of Squid and hrp38 proteins inhibits splicing of the intron in the Hsr omega-RC transcript, but enhances splicing of the intron in the Ddc pre-mRNA. Taken together, these findings suggest that poly(ADP-ribosyl)ation regulates the interaction between hnRNPs and RNA and thus modulates the splicing pathways.
The domestic pig is an important "dual purpose" animal model for agricultural and biomedical applications. There is an emerging consensus in the biomedical community for the use of large animal models such as pigs to either serve as an alternative, or complement investigations from the mouse. However, the use of pig has not proven popular due to technical difficulties and time required in generating models with desired genetic modifications. In this regard, the ability to directly modify the genome in the zygote and generate edited animals is highly desirable. This report demonstrates for the first time, the generation of gene targeted animals by direct injection of Cas9 ribonucleoprotein complex and short stretches of DNA sequences into porcine zygotes. The Cas9 protein from Streptococcus pyogenes was pre-complexed with a single guide RNA targeting downstream of the ubiquitously expressed COL1A gene, and co-injected with a single-stranded repair template into porcine zygotes. Using this approach a line of pigs that carry pseudo attP sites within the COL1A locus to enable phiC31 integrase mediated introduction of transgenes has been generated. This new route for genome engineering in pigs via zygote injection should greatly enhance applications in both agriculture and biomedicine.
Ribonucleoprotein (RNP) complex-mediated base editing is expected to be greatly beneficial because of its reduced off-target effects compared to plasmid- or viral vector-mediated gene editing, especially in therapeutic applications. However, production of recombinant cytosine base editors (CBEs) or adenine base editors (ABEs) with ample yield and high purity in bacterial systems is challenging. Here, we obtained highly purified CBE/ABE proteins from a human cell expression system and showed that CBE/ABE RNPs exhibited different editing patterns (i.e., less conversion ratio of multiple bases to single base) compared to plasmid-encoded CBE/ABE, mainly because of the limited life span of RNPs in cells. Furthermore, we found that off-target effects in both DNA and RNA were greatly reduced for ABE RNPs compared to plasmid-encoded ABE. We ultimately applied NG PAM-targetable ABE RNPs to in vivo gene correction in retinal degeneration 12 (rd12) model mice.
Natural killer (NK) cells are an attractive cell-type for adoptive immunotherapy, but challenges in preparation of therapeutic primary NK cells restrict patient accessibility to NK cell immunotherapy. NK-92 is a well-characterized human NK cell line that has demonstrated promising anti-cancer activities in clinical trials. Unlimited proliferation of NK-92 cells provides a consistent supply of cells for the administration and development of NK cell immunotherapy. However, the clinical efficacy of NK-92 cells has not reached its full potential due to reduced immune functions as compared to primary NK cells. Improvements of NK-92 functions currently rely on conventional transgene delivery by mRNA, plasmid and viral vector with limited efficiencies. To enable precise genetic modifications, we have established a robust CRISPR genome engineering platform for NK-92 based on the nucleofection of Cas9 ribonucleoprotein. To demonstrate the versatility of the platform, we have performed cell-based screening of Cas9 guide RNA, multiplex gene knockout of activating and inhibitory receptors, knock-in of a fluorescent gene, and promoter insertion to reactivate endogenous CD16 and DNAM-1. The CRISPR-engineered NK-92 demonstrated markedly enhanced cytotoxicity and could mediate antibody-dependent cellular cytotoxicity against hard to kill cancer cell lines. Our genome editing platform is straightforward and robust for both functional studies and therapeutic engineering of NK-92 cells.
Messenger ribonucleoprotein particles (mRNPs) are used to transport mRNAs along neuronal dendrites to their site of translation. Numerous mRNA-binding and regulatory proteins within mRNPs finely regulate the fate of bound-mRNAs. Their specific combination defines different types of mRNPs that in turn are related to specific synaptic functions. One of these mRNA-binding proteins, Staufen2 (Stau2), was shown to transport dendritic mRNAs along microtubules. Its knockdown expression in neurons was shown to change spine morphology and synaptic functions. To further understand the molecular mechanisms by which Stau2 modulates synaptic function in neurons, it is important to identify and characterize protein co-factors that regulate the fate of Stau2-containing mRNPs. To this end, a proteomic approach was used to identify co-immunoprecipitated proteins in Staufen2-containing mRNPs isolated from embryonic rat brains. The proteomic approach identified mRNA-binding proteins (PABPC1, hnRNP H1, YB1 and hsc70), proteins of the cytoskeleton (alpha- and beta-tubulin) and RUFY3 a poorly characterized protein. While PABPC1 and YB1 associate with Stau2-containing mRNPs through RNAs, hsc70 is directly bound to Stau2 and this interaction is regulated by ATP. PABPC1 and YB1 proteins formed puncta in dendrites of embryonic rat hippocampal neurons. However, they poorly co-localized with Stau2 in the large dendritic complexes suggesting that they are rather components of Stau2-containing mRNA particles. All together, these results represent a further step in the characterization of Stau2-containing mRNPs in neurons and provide new tools to study and understand how Stau2-containing mRNPs are transported, translationally silenced during transport and/or locally expressed according to cell needs.
Post-transcriptional control is nowadays considered a main checking point for correct gene regulation during development, and RNA binding proteins actively participate in this process. Arabidopsis thaliana FLOWERING LOCUS WITH KH DOMAINS (FLK) and PEPPER (PEP) genes encode RNA-binding proteins that contain three K-homology (KH)-domain, the typical configuration of Poly(C)-binding ribonucleoproteins (PCBPs). We previously demonstrated that FLK and PEP interact to regulate FLOWERING LOCUS C (FLC), a central repressor of flowering time. Now we show that FLK and PEP also play an important role in the maintenance of the C-function during floral organ identity by post-transcriptionally regulating the MADS-box floral homeotic gene AGAMOUS (AG). Previous studies have indicated that the KH-domain containing protein HEN4, in concert with the CCCH-type RNA binding protein HUA1 and the RPR-type protein HUA2, facilitates maturation of the AG pre-mRNA. In this report we show that FLK and PEP genetically interact with HEN4, HUA1, and HUA2, and that the FLK and PEP proteins physically associate with HUA1 and HEN4. Taken together, these data suggest that HUA1, HEN4, PEP and FLK are components of the same post-transcriptional regulatory module that ensures normal processing of the AG pre-mRNA. Our data better delineates the roles of PEP in plant development and, for the first time, links FLK to a morphogenetic process.
Monitoring mitochondrial function is crucial for organismal survival. This task is performed by mitochondrial surveillance or quality control pathways, which are activated by signals originating from mitochondria and relayed to the nucleus (retrograde response) to start transcription of protective genes. In Caenorhabditis elegans, several systems are known to play this role, including the UPRmt, MAPKmt, and the ESRE pathways. These pathways are highly conserved and their loss compromises survival following mitochondrial stress. In this study, we found a novel interaction between the box C/D snoRNA core proteins (snoRNPs) and mitochondrial surveillance and innate immune pathways. We showed that box C/D, but not box H/ACA, snoRNPs are required for the full function of UPRmt and ESRE upon stress. The loss of box C/D snoRNPs reduced mitochondrial mass, mitochondrial membrane potential, and oxygen consumption rate, indicating overall degradation of mitochondrial function. Concomitantly, the loss of C/D snoRNPs increased immune response and reduced host intestinal colonization by infectious bacteria, improving host resistance to pathogenesis. Our data may indicate a model wherein box C/D snoRNP machinery regulates a "switch" of the cell's activity between mitochondrial surveillance and innate immune activation. Understanding this mechanism is likely to be important for understanding multifactorial processes, including responses to infection and aging.
The pig is an ideal large animal model for genetic engineering applications. A relatively short gestation interval and large litter size makes the pig a conducive model for generating and propagating genetic modifications. The domestic pig also shares close similarity in anatomy, physiology, size, and life expectancy, making it an ideal animal for modeling human diseases. Often, however, the technical difficulties in generating desired genetic modifications such as targeted knockin of short stretches of sequences or transgenes have impeded progress in this field. In this study, we have investigated and compared the relative efficiency of CRISPR/Cas ribonucleoproteins in engineering targeted knockin of pseudo attP sites downstream of a ubiquitously expressed COL1A gene in porcine somatic cells and generated live fetuses by somatic cell nuclear transfer (SCNT). By leveraging these knockin pseudo attP sites, we have demonstrated subsequent phiC31 integrase mediated integration of green fluorescent protein (GFP) transgene into the site. This work for the first time created an optimized protocol for CRISPR/Cas mediated knockin in porcine somatic cells, while simultaneously creating a stable platform for future transgene integration and generating transgenic animals.
Messenger RNA processing in trypanosomes by cis and trans splicing requires spliceosomal small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/U6, and U5, as well as the spliced leader (SL) RNP. As in other eukaryotes, these RNPs share a core structure of seven Sm polypeptides. Here, we report that the identity of the Sm protein constituents varies between spliceosomal snRNPs: specifically, two of the canonical Sm proteins, SmB and SmD3, are replaced in the U2 snRNP by two novel, U2 snRNP-specific Sm proteins, Sm15K and Sm16.5K. We present a model for the variant Sm core in the U2 snRNP, based on tandem affinity purification-tagging and in vitro protein-protein interaction assays. Using in vitro reconstitutions with canonical and U2-specific Sm cores, we show that the exchange of two Sm subunits determines discrimination between individual Sm sites. In sum, we have demonstrated that the heteroheptameric Sm core structure varies between spliceosomal snRNPs, and that modulation of the Sm core composition mediates the recognition of small nuclear RNA-specific Sm sites.
Many ribonucleoproteins (RNPs), which are comprised of noncoding RNA and associated proteins, are involved in essential cellular processes such as translation and pre-mRNA splicing. One class of RNP is the small Cajal body-specific RNP (scaRNP), which contributes to the biogenesis of small nuclear RNPs (snRNPs) that are central components of the spliceosome. Three scaRNAs are internally processed, generating stable nucleolus-enriched RNAs of unknown function. Here, we provide data that show that these RNAs become part of RNPs we term regulatory RNPs (regRNPs). Most modifications within rRNA (predominantly pseudouridylation and ribose 2'-O-methylation) are conducted by small nucleolar RNPs (snoRNPs), and we provide evidence that the activity of at least some of these snoRNPs is under the control of regRNPs. Because modifications within rRNA can vary in different physiological or pathological situations, rRNA modifications are thought to be the major source of ribosome heterogeneity. Our identification of regRNPs thus provides a potential mechanism for how ribosome heterogeneity may be accomplished. This work also provides additional functional connections between the Cajal body and the nucleolus.
The A/B subfamily of heterogeneous nuclear ribonucleoproteins (hnRNPs A/B), which includes hnRNP A1, A2/B1, and A3, plays an important role in cell proliferation. The simultaneous suppression of hnRNP A1/A2, but not the suppression of hnRNP A1 or A2 alone, has been shown to inhibit cell proliferation and induce apoptosis in cancer cells, but not in mortal normal cells. However, the molecular basis for such a differential inhibition of cell proliferation remains unknown. Here, we show that the simultaneous suppression of hnRNP A1 and hnRNP A2 resulted in dysfunctional telomeres and induced DNA damage responses in cancer cells. The inhibition of apoptosis did not alleviate the inhibition of cell proliferation nor the formation of dysfunctional telomeres in cancer cells depleted of hnRNP A1/A2. Moreover, while proliferation of mortal normal fibroblasts was not sensitive to the depletion of hnRNP A1/A2, the ectopic expression of hTERT in normal fibroblasts rendered these cells sensitive to proliferation inhibition, which was associated with the production of dysfunctional telomeres. Our study demonstrates that hnRNP A1 and A2 function to maintain telomeres in telomerase-expressing cells only, suggesting that the maintenance of functional telomeres in telomerase-expressing cancer cells employs factors that differ from those used in the telomerase-negative normal cells.
Frontotemporal lobar degeneration (FTLD) is pathologically subdivided based on the presence of particular pathological proteins that are identified in inclusion bodies observed post-mortem. The FTLD-FUS subgroup is defined by the presence of the fused in sarcoma protein (FUS) in pathological inclusions. FUS is a heterogeneous nuclear ribonucleoprotein (hnRNP) protein and a member of the FET (FUS, EWS, TAF15) protein family. It shuttles between the nucleus and cytoplasm, and has been implicated in many cellular functions including translation, splicing, and RNA transport. EWS, TAF15 and the nuclear import receptor transportin have been shown to co-accumulate with FUS in neuronal inclusions specifically in FTLD-FUS, with transportin-positive inclusions most frequently observed. Here, we report the identification of hnRNP R and hnRNP Q in neuronal cytoplasmic and intranuclear inclusions in the frontal cortex and hippocampus of FTLD-FUS patients, as frequently as transportin. hnRNP R and hnRNP Q were not found in the characteristic pathological inclusions observed in FTLD-TDP (subtypes A-C). Additionally, we studied the expression of hnRNP R in the frontal and temporal cortices from patients with FTLD and found significantly increased expression of the heterogeneous nuclear ribonucleoprotein R in several FTLD disease groups. Our identification of the frequent presence of hnRNP R and hnRNP Q in FTLD-FUS inclusions suggests a potential role for these hnRNPs in FTLD-FUS pathogenesis and supports the role of dysfunctional RNA metabolism in FTLD.
Influenza A virus has an eight-partite RNA genome that during viral assembly forms a complex containing one copy of each RNA. Genome assembly is a selective process driven by RNA-RNA interactions and is hypothesized to lead to discrete punctate structures scattered through the cytosol. Here, we show that contrary to the accepted view, formation of these structures precedes RNA-RNA interactions among distinct viral ribonucleoproteins (vRNPs), as they assemble in cells expressing only one vRNP type. We demonstrate that these viral inclusions display characteristics of liquid organelles, segregating from the cytosol without a delimitating membrane, dynamically exchanging material and adapting fast to environmental changes. We provide evidence that viral inclusions develop close to endoplasmic reticulum (ER) exit sites, depend on continuous ER-Golgi vesicular cycling and do not promote escape to interferon response. We propose that viral inclusions segregate vRNPs from the cytosol and facilitate selected RNA-RNA interactions in a liquid environment.
Respiratory syncytial virus (RSV) is the primary cause of severe respiratory infection in infants worldwide. Replication of RSV genomic RNA occurs in cytoplasmic inclusions generating viral ribonucleoprotein complexes (vRNPs). vRNPs then reach assembly and budding sites at the plasma membrane. However, mechanisms ensuring vRNPs transportation are unknown. We generated a recombinant RSV harboring fluorescent RNPs allowing us to visualize moving vRNPs in living infected cells and developed an automated imaging pipeline to characterize the movements of vRNPs at a high throughput. Automatic tracking of vRNPs revealed that around 10% of the RNPs exhibit fast and directed motion compatible with transport along the microtubules. Visualization of vRNPs moving along labeled microtubules and restriction of their movements by microtubule depolymerization further support microtubules involvement in vRNPs trafficking. Approximately 30% of vRNPs colocalize with Rab11a protein, a marker of the endosome recycling (ER) pathway and we observed vRNPs and Rab11-labeled vesicles moving together. Transient inhibition of Rab11a expression significantly reduces vRNPs movements demonstrating Rab11 involvement in RNPs trafficking. Finally, Rab11a is specifically immunoprecipitated with vRNPs in infected cells suggesting an interaction between Rab11 and the vRNPs. Altogether, our results strongly suggest that RSV RNPs move on microtubules by hijacking the ER pathway.
Telomerase is the ribonucleoprotein enzyme that replenishes telomeric DNA and maintains genome integrity. Minimally, telomerase activity requires a templating RNA and a catalytic protein. Additional proteins are required for activity on telomeres in vivo. Here, we report that the Pop1, Pop6, and Pop7 proteins, known components of RNase P and RNase MRP, bind to yeast telomerase RNA and are essential constituents of the telomerase holoenzyme. Pop1/Pop6/Pop7 binding is specific and involves an RNA domain highly similar to a protein-binding domain in the RNAs of RNase P/MRP. The results also show that Pop1/Pop6/Pop7 function to maintain the essential components Est1 and Est2 on the RNA in vivo. Consistently, addition of Pop1 allows for telomerase activity reconstitution with wild-type telomerase RNA in vitro. Thus, the same chaperoning module has allowed the evolution of functionally and, remarkably, structurally distinct RNPs, telomerase, and RNases P/MRP from unrelated progenitor RNAs.
Small nuclear ribonucleoproteins (snRNPs) are core components of the spliceosome. The U1, U2, U4, and U5 snRNPs each contain a common set of seven Sm proteins. Three of these Sm proteins are posttranslationally modified to contain symmetric dimethylarginine (sDMA) residues within their C-terminal tails. However, the precise function of this modification in the snRNP biogenesis pathway is unclear. Several lines of evidence suggest that the methyltransferase protein arginine methyltransferase 5 (PRMT5) is responsible for sDMA modification of Sm proteins. We found that in human cells, PRMT5 and a newly discovered type II methyltransferase, PRMT7, are each required for Sm protein sDMA modification. Furthermore, we show that the two enzymes function nonredundantly in Sm protein methylation. Lastly, we provide in vivo evidence demonstrating that Sm protein sDMA modification is required for snRNP biogenesis in human cells.
Microalgae are versatile organisms capable of converting CO2, H2O, and sunlight into fuel and chemicals for domestic and industrial consumption. Thus, genetic modifications of microalgae for enhancing photosynthetic productivity, and biomass and bio-products generation are crucial for both academic and industrial applications. However, targeted mutagenesis in microalgae with CRISPR-Cas9 is limited. Here we report, a one-step transformation of Chlamydomonas reinhardtii by the DNA-free CRISPR-Cas9 method rather than plasmids that encode Cas9 and guide RNAs. Outcome was the sequential CpFTSY and ZEP two-gene knockout and the generation of a strain constitutively producing zeaxanthin and showing improved photosynthetic productivity.
Genome engineering of primary human cells with CRISPR-Cas9 has revolutionized experimental and therapeutic approaches to cell biology, but human myeloid-lineage cells have remained largely genetically intractable. We present a method for the delivery of CRISPR-Cas9 ribonucleoprotein (RNP) complexes by nucleofection directly into CD14+ human monocytes purified from peripheral blood, leading to high rates of precise gene knockout. These cells can be efficiently differentiated into monocyte-derived macrophages or dendritic cells. This process yields genetically edited cells that retain transcript and protein markers of myeloid differentiation and phagocytic function. Genetic ablation of the restriction factor SAMHD1 increased HIV-1 infection >50-fold, demonstrating the power of this system for genotype-phenotype interrogation. This fast, flexible, and scalable platform can be used for genetic studies of human myeloid cells in immune signaling, inflammation, cancer immunology, host-pathogen interactions, and beyond, and could facilitate the development of myeloid cellular therapies.
Welcome to the FDI Lab - SciCrunch.org Resources search. From here you can search through a compilation of resources used by FDI Lab - SciCrunch.org and see how data is organized within our community.
You are currently on the Community Resources tab looking through categories and sources that FDI Lab - SciCrunch.org has compiled. You can navigate through those categories from here or change to a different tab to execute your search through. Each tab gives a different perspective on data.
If you have an account on FDI Lab - SciCrunch.org then you can log in from here to get additional features in FDI Lab - SciCrunch.org such as Collections, Saved Searches, and managing Resources.
Here is the search term that is being executed, you can type in anything you want to search for. Some tips to help searching:
You can save any searches you perform for quick access to later from here.
We recognized your search term and included synonyms and inferred terms along side your term to help get the data you are looking for.
If you are logged into FDI Lab - SciCrunch.org you can add data records to your collections to create custom spreadsheets across multiple sources of data.
Here are the facets that you can filter your papers by.
From here we'll present any options for the literature, such as exporting your current results.
If you have any further questions please check out our FAQs Page to ask questions and see our tutorials. Click this button to view this tutorial again.
Year:
Count: