CD32-RNA Co-localizes with HIV-RNA in CD3+ Cells Found within Gut Tissues from Viremic and ART-Suppressed Individuals.

BackgroundIdentifying biomarkers for cells harboring replication-competent HIV is a major research priority. Recently, there have been mixed reports addressing the possibility that CD32-expressing T cells are enriched for HIV. There is growing evidence that CD32 expression increases with cellular activation that may be related to, but not necessarily specific for, infection with HIV. However, the relationship of CD32 expression to HIV-infection in subtypes of tissue-resident leukocytes is unclear.MethodsFirst, we used duplex chromogenic in situ hybridization to identify cells actively transcribing RNA for both CD32 and HIV on human gut tissues. Then we performed multiplexed immunofluorescence and in situ hybridization (mIFISH) on sections from the same tissues to determine the phenotype of individual cells co-expressing HIV-RNA and CD32-RNA.ResultsHIV-RNA+ cells were more abundant in tissues from viremic individuals than in those receiving suppressive anti-retroviral therapy (ART). However, staining by both methods indicated that a higher proportion of HIV-RNA+ cells co-expressed CD32-RNA in ART-suppressed individuals than in those with viremia. The majority of HIV-RNA+ cells were CD3+.ConclusionsOur data suggest that the transcription of CD32-RNA is correlated with HIV transcriptional activity in CD3+ cells found within human gut tissue. Whether or not up-regulation of CD32-RNA is a direct result of HIV transcription or more global T-cell activation remains unclear

Visualization of positive and negative sense viral RNA for probing the mechanism of direct-acting antivirals against hepatitis C virus

RNA viruses are highly successful pathogens and are the causative agents for many important diseases. To fully understand the replication of these viruses it is necessary to address the roles of both positive-strand RNA ((+)RNA) and negative-strand RNA ((-)RNA), and their interplay with viral and host proteins. Here we used branched DNA (bDNA) fluorescence in situ hybridization (FISH) to stain both the abundant (+)RNA and the far less abundant (-)RNA in both hepatitis C virus (HCV)- and Zika virus-infected cells, and combined these analyses with visualization of viral proteins through confocal imaging. We were able to phenotypically examine HCV-infected cells in the presence of uninfected cells and revealed the effect of direct-acting antivirals on HCV (+)RNA, (-)RNA, and protein, within hours of commencing treatment. Herein, we demonstrate that bDNA FISH is a powerful tool for the study of RNA viruses that can provide insights into drug efficacy and mechanism of action

tRNA Profiling of Mesenchymal Stem Cell Exosome

Background: Exosomes have great potential in regenerative medicine through the transfer of their bioactive cargos, such as RNA. tRF RNA and tiRNA are tRNAderived non-coding RNA. Here, we sought to identify the tRF/tiRNA profile in human mesenchymal stem cell (hMSC) exosomes. Methods: Bone marrow hMSCs were cultured with/without osteogenic differentiation medium and exosomes were harvested. RNA was extracted from: 1) control cells (Cell-NT); 2) control exosomes (EXO-NT); 3) differentiated cells (Cell-OM); 4) exosomes produced by differentiated cells (EXO-OM). RNA was sequenced to profile the small RNA with a focus on tRF/tiRNA. Results: tRF/tiRNA was highly enriched in hMSC exosomes. Less diversity was seen in the tRF/tiRNA profile in exosomes than that in parent cells. Selective tRF/tiRNA were packed into MSC exosomes and their profile is dependent on the cell maturation status. Conclusions: Our results suggest that tRF/tiRNA may play a role in mediating the function of exosomes in tissue regeneration

Primary osteoblast-like cells from patients with end-stage kidney disease reflect gene expression, proliferation, and mineralization characteristics ex vivo.

Osteocytes regulate bone turnover and mineralization in chronic kidney disease. As osteocytes are derived from osteoblasts, alterations in osteoblast function may regulate osteoblast maturation, osteocytic transition, bone turnover, and skeletal mineralization. Thus, primary osteoblast-like cells were cultured from bone chips obtained from 24 pediatric ESKD patients. RNA expression in cultured cells was compared with RNA expression in cells from healthy individuals, to RNA expression in the bone core itself, and to parameters of bone histomorphometry. Proliferation and mineralization rates of patient cells were compared with rates in healthy control cells. Associations were observed between bone osteoid accumulation, as assessed by bone histomorphometry, and bone core RNA expression of osterix, matrix gla protein, parathyroid hormone receptor 1, and RANKL. Gene expression of osteoblast markers was increased in cells from ESKD patients and signaling genes including Cyp24A1, Cyp27B1, VDR, and NHERF1 correlated between cells and bone cores. Cells from patients with high turnover renal osteodystrophy proliferated more rapidly and mineralized more slowly than did cells from healthy controls. Thus, primary osteoblasts obtained from patients with ESKD retain changes in gene expression ex vivo that are also observed in bone core specimens. Evaluation of these cells in vitro may provide further insights into the abnormal bone biology that persists, despite current therapies, in patients with ESKD

Techniques for RNA in vivo imaging in plants

Since the discovery of small RNAs and RNA silencing, RNA biology has taken a centre stage in cell and developmental biology. Small RNAs, but also mRNAs and other types of cellular and viral RNAs are processed at specific subcellular localizations. To fully understand cellular RNA metabolism and the various processes influenced byit, techniques are required that permit the sequence-specific tracking of RNAs in living cells. A variety of methods for RNA visualization have been developed since the 1990s, but plant cells pose particular challenges and not all approaches are applicable to them. On the other hand, plant RNA metabolism is particularly diverse and RNAs are even transported between cells, so RNA imaging can potentially provide many valuable insights into plant function at the cellular and tissue level. This Short Review briefly introduces the currently available techniques for plant RNA in vivo imaging and discusses their suitability for different biological questions.PostprintPeer reviewe

Delayed kinetics of poliovirus RNA synthesis in a human cell line with reduced levels of hnRNP C proteins.

The hnRNP C heterotetramer [(C1(3))C2] binds RNA polymerase II transcripts in the nucleus, along with other proteins of the core hnRNP complex, and plays an important role in mRNA biogenesis and transport. Infection of HeLa cells with poliovirus causes hnRNP C to re-localize from the nucleus, where it is normally retained during interphase, to the cytoplasm. We have proposed that in the cytoplasm, the protein isoforms of hnRNP C participate in the recognition of viral specific RNAs by the poliovirus replication proteins and/or in the assembly of membrane-bound RNA replication complexes. In SK-OV-3 cells, which express reduced levels of hnRNP C compared to HeLa cells or 293 cells, the kinetics of poliovirus replication are delayed. hnRNP C is also re-localized from the nucleus to the cytoplasm in SK-OV-3 cells infected with poliovirus. Increased expression of hnRNP C in SK-OV-3 cells by transient transfection increases the rate of virus production and overall yield over that seen in mock-transfected cells. We propose that hnRNP C interacts with poliovirus RNA and replication proteins to increase the efficiency of viral genomic RNA synthesis

FLASH: ultra-fast protocol to identify RNA-protein interactions in cells

Determination of the in vivo binding sites of RNA-binding proteins (RBPs) is paramount to understanding their function and how they affect different aspects of gene regulation. With hundreds of RNA-binding proteins identified in human cells, a flexible, high-resolution, high-throughput, highly multiplexible and radioactivity-free method to determine their binding sites has not been described to date. Here we report FLASH (Fast Ligation of RNA after some sort of Affinity Purification for High-throughput Sequencing), which uses a special adapter design and an optimized protocol to determine protein-RNA interactions in living cells. The entire FLASH protocol, starting from cells on plates to a sequencing library, takes 1.5 days. We demonstrate the flexibility, speed and versatility of FLASH by using it to determine RNA targets of both tagged and endogenously expressed proteins under diverse conditions in vivo

Gene expression and splicing alterations analyzed by high throughput RNA sequencing of chronic lymphocytic leukemia specimens.

BackgroundTo determine differentially expressed and spliced RNA transcripts in chronic lymphocytic leukemia specimens a high throughput RNA-sequencing (HTS RNA-seq) analysis was performed.MethodsTen CLL specimens and five normal peripheral blood CD19+ B cells were analyzed by HTS RNA-seq. The library preparation was performed with Illumina TrueSeq RNA kit and analyzed by Illumina HiSeq 2000 sequencing system.ResultsAn average of 48.5 million reads for B cells, and 50.6 million reads for CLL specimens were obtained with 10396 and 10448 assembled transcripts for normal B cells and primary CLL specimens respectively. With the Cuffdiff analysis, 2091 differentially expressed genes (DEG) between B cells and CLL specimens based on FPKM (fragments per kilobase of transcript per million reads and false discovery rate, FDR q < 0.05, fold change >2) were identified. Expression of selected DEGs (n = 32) with up regulated and down regulated expression in CLL from RNA-seq data were also analyzed by qRT-PCR in a test cohort of CLL specimens. Even though there was a variation in fold expression of DEG genes between RNA-seq and qRT-PCR; more than 90 % of analyzed genes were validated by qRT-PCR analysis. Analysis of RNA-seq data for splicing alterations in CLL and B cells was performed by Multivariate Analysis of Transcript Splicing (MATS analysis). Skipped exon was the most frequent splicing alteration in CLL specimens with 128 significant events (P-value <0.05, minimum inclusion level difference >0.1).ConclusionThe RNA-seq analysis of CLL specimens identifies novel DEG and alternatively spliced genes that are potential prognostic markers and therapeutic targets. High level of validation by qRT-PCR for a number of DEG genes supports the accuracy of this analysis. Global comparison of transcriptomes of B cells, IGVH non-mutated CLL (U-CLL) and mutated CLL specimens (M-CLL) with multidimensional scaling analysis was able to segregate CLL and B cell transcriptomes but the M-CLL and U-CLL transcriptomes were indistinguishable. The analysis of HTS RNA-seq data to identify alternative splicing events and other genetic abnormalities specific to CLL is an added advantage of RNA-seq that is not feasible with other genome wide analysis

Detection of DNA-RNA hybrids in vivo

DNA-RNA hybrids form naturally during essential cellular functions such as transcription and replication. However, they may be an important source of genome instability, a hallmark of cancer and genetic diseases. Detection of DNA-RNA hybrids in cells is becoming crucial to understand an increasing number of molecular biology processes in genome dynamics and function and to identify new factors and mechanisms responsible for disease in biomedical research. Here, we describe two different procedures for the reliable detection of DNA-RNA hybrids in the yeast Saccharomyces cerevisiae and in human cells: DNA-RNA Immunoprecipitation (DRIP) and Immunofluorescence