Browsing by Subject "RNA sequencing"

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Definition of the yeast transcriptome using next-generation RNA sequencing
(2012-05) Fairchild, Lauren; Iyer, Vishwanath
RNA sequencing (RNA-seq) is a high-throughput method by which the sequence of each RNA molecule in an organism can be determined. Because of its high-throughput nature and high accuracy, RNA-seq allows for the study of the transcriptome at base-pair resolution and for the discovery of novel transcripts and splice junctions. Although the transcriptome of Saccharomyces cerevisiae (baker’s yeast) has been previously studied, the use of RNA-seq allows for the characterization of novel and low abundance transcripts that are difficult to study using more traditional methods. A robust library preparation is necessary to take advantage of the strengths of RNA-seq. In order to find an appropriate library preparation, several protocols were prototyped and the results of these preparations were compared against similar expression microarray experiments. qPCR was used to validate the RNA abundance of a selection of genes with a known response to the treatment being used. After several iterations, a library preparation was found which reliably reproduced the microarray results while offering additional information such as the transcription start site and transcript architecture. However, this preparation did not preserve the directionality of the RNA transcripts. Strand direction information is useful when assigning sequenced reads to transcripts and when differentiating signal from noise. A new library preparation is being prototyped which will preserve the directionality of the transcripts, allowing for an additional dimension of information to be captured by the data. The yeast response to various perturbations including heat shock and sporulation will be characterized using this method. By preserving the directionality of the RNA transcripts and by studying yeast under several conditions, the yeast transcriptome will be defined at a higher resolution.
A Human Torque Teno Virus Encodes a MicroRNA That Inhibits Interferon Signaling
(PLOS Pathogens, 2013-12-19) Kincaid, Rodney P.; Burke, James M.; Cox, Jennifer C.; de Villiers, Ethel-Michele; Sullivan, Christopher S.
Torque teno viruses (TTVs) are a group of viruses with small, circular DNA genomes. Members of this family are thought to ubiquitously infect humans, although causal disease associations are currently lacking. At present, there is no understanding of how infection with this diverse group of viruses is so prevalent. Using a combined computational and synthetic approach, we predict and identify miRNA-coding regions in diverse human TTVs and provide evidence for TTV miRNA production in vivo. The TTV miRNAs are transcribed by RNA polymerase II, processed by Drosha and Dicer, and are active in RISC. A TTV mutant defective for miRNA production replicates as well as wild type virus genome; demonstrating that the TTV miRNA is dispensable for genome replication in a cell culture model. We demonstrate that a recombinant TTV genome is capable of expressing an exogenous miRNA, indicating the potential utility of TTV as a small RNA vector. Gene expression profiling of host cells identifies N-myc (and STAT) interactor (NMI) as a target of a TTV miRNA. NMI transcripts are directly regulated through a binding site in the 3′UTR. SiRNA knockdown of NMI contributes to a decreased response to interferon signaling. Consistent with this, we show that a TTV miRNA mediates a decreased response to IFN and increased cellular proliferation in the presence of IFN. Thus, we add Annelloviridae to the growing list of virus families that encode miRNAs, and suggest that miRNA-mediated immune evasion can contribute to the pervasiveness associated with some of these viruses.
Immunosuppressive effects of PEG-fusion in peripheral nerve allografts
(2021-03-08) Smith, Tyler Aaron; Bittner, George D.; Tucker, Haley O.; Sakiyama-Elbert, Shelly E.; Zakon, Harold; Poenie, Martin
Surgical repair of ablation-type peripheral nerve injuries by using peripheral nerve allografts (PNAs) has been hindered for decades due to slow and ineffective axon regeneration from proximal nerve ends as well as immunological rejection of PNAs. We have developed a polyethylene glycol (PEG)-fusion repair protocol for sciatic PNAs in rats that results in maintenance of myelinated axons that do not degenerate, neuromuscular junction innervation, and significantly improved behavioral recovery, as compared to current nerve repair methods. These phenotypes are maintained for weeks postoperatively without using tissue-matching, decellularization, or immunosuppressive drugs. That is, PEG-fused PNAs are functionally tolerated by the host immune system. This dissertation presents work performed to characterize and investigate mechanisms underlying immunological responses to PNAs treated with PEG-fusion with the aim of understanding how and why PEG-fused allografts are not rejected by the host immune system in rat sciatic nerve injury models. Chapter 1 provides a review of the clinical significance of peripheral nerve injury, including: Biological processes of axonal degeneration and regeneration, current methods of nerve repair, immunological rejection of PNAs, concepts underlying PEG-fusion repair and current experimental results of PEG-fusion in rat PNAs, as well as the hypotheses and aims explored. Chapter 2 characterizes innate and adaptive immune responses to PEG-fused PNAs using primarily immunohistochemistry, electron microscopy, and quantitative reverse transcription PCR (RT-qPCR). Our results suggest that PEG-fused PNAs achieve immunotolerance via attenuated innate and adaptive immune responses. Chapter 3 examines via RNA sequencing the coding transcriptome of PEG-fused PNAs to determine which biological processes, protein families, pathways, and protein-protein interaction networks differentiate PEG-fused PNAs from negative control PNAs not treated with PEG with the goal of identifying potential mechanisms underlying immunotolerance. This work provides a critical molecular foundation for future studies investigating PEG-fusion-mediated immunosuppression in PNAs. Chapter 4 investigates whether treatment of PNAs with PEG alone without axonal fusion induces similar immunosuppressive effects. Our results suggest that PEG treatment alone does not prevent Wallerian degeneration or attenuation of innate and adaptive immune responses. Chapter 5 provides a summary of the dissertation work and describes future directions for research.
The Retrohoming of Linear Group II Intron RNAs in Drosophila melanogaster Occurs by Both DNA Ligase 4–Dependent and –Independent Mechanisms
(Public Library of Science, 2012-02-16) White, Travis B.; Lambowitz, Alan M.
Mobile group II introns are bacterial retrotransposons that are thought to have invaded early eukaryotes and evolved into introns and retroelements in higher organisms. In bacteria, group II introns typically retrohome via full reverse splicing of an excised intron lariat RNA into a DNA site, where it is reverse transcribed by the intron-encoded protein. Recently, we showed that linear group II intron RNAs, which can result from hydrolytic splicing or debranching of lariat RNAs, can retrohome in eukaryotes by performing only the first step of reverse splicing, ligating their 3′ end to the downstream DNA exon. Reverse transcription then yields an intron cDNA, whose free end is linked to the upstream DNA exon by an error-prone process that yields junctions similar to those formed by non-homologous end joining (NHEJ). Here, by using Drosophila melanogaster NHEJ mutants, we show that linear intron RNA retrohoming occurs by major Lig4-dependent and minor Lig4-independent mechanisms, which appear to be related to classical and alternate NHEJ, respectively. The DNA repair polymerase θ plays a crucial role in both pathways. Surprisingly, however, mutations in Ku70, which functions in capping chromosome ends during NHEJ, have only moderate, possibly indirect effects, suggesting that both Lig4 and the alternate end-joining ligase act in some retrohoming events independently of Ku. Another potential Lig4-independent mechanism, reverse transcriptase template switching from the intron RNA to the upstream exon DNA, occurs in vitro, but gives junctions differing from the majority in vivo. Our results show that group II introns can utilize cellular NHEJ enzymes for retromobility in higher organisms, possibly exploiting mechanisms that contribute to retrotransposition and mitigate DNA damage by resident retrotransposons. Additionally, our results reveal novel activities of group II intron reverse transcriptases, with implications for retrohoming mechanisms and potential biotechnological applications.
Small RNA Discovery Using Conservation Analysis Enhancement
(2016-04-19) Chou, Brendan; Contreras, Lydia M.
Small noncoding RNAs (sRNAs) have been described as posttranscriptional regulators of many cellular systems in bacteria, notably environmental stress responses. However, sRNAs are small and difficult to detect due to the lack of uniformity in which they are encoded in the genome. Since they may be found inn the 5' and 3' untranslated regions of genes and in intergenic regions, identification and characterization of sRNAs has been challenging. These elusive gene expression regulators may aid in explaining the interesting survival mechanisms of Deinococcus radiodurans under ionizing radation of > 12,000 Gy, while most bacteria find 1,000 Gy of radiation to be lethal. This study takes advantage of high-throughput experimental methods and computational prediction methods to develop a pipeline for more efficient sRNA discovery. Intergenic regions in 13 well-annotated bacterial species were analyzed for conservation levels to increase the likelihood that candidate intergenic regions would code for sRNAs. In total, more than 900 validated bacterial sRNAs and 23,000 intergenic regions were analyzed. The results indicated that sRNAs are found more abundantly in longer and highly conserved intergenic regions. This workflow was used to discover novel sRNA in D. radiodurans. 199 potential sRNA candidates were generated from whole-transcriptome deep sequencing analysis, and 41 were confirmed using northern blotting and reverse transcription PCR. Analysis of sRNA expression levels during recovery after actute radiation resulted in 8 confirmed sRNAs which demonstrated varying expression levels due to irradiation. Conservation comparison of novel D. radiodurans sRNAs led to the discovery and confirmation of 7 homologous sRNAs in the closely-related species Deinococcus geothermalis.
Transmembrane stem cell factor protein therapeutics enhance revascularization in ischemia without mast cell activation
(2020-05-07) Takematsu, Eri; Baker, Aaron Blair; Dunn, Andrew K.; Terreson, David W.; Stachowiak , Jeanne C.; Suggs, Laura J.
Diabetes mellitus affects approximately 350 million people worldwide, leading to the death of about 4.6 million people per year. As a complication of diabetes, 30-40 percent of patients age 50 and older develop peripheral artery disease (PAD). The current standard of care treatments for PAD includes surgical revascularization with bypass grafting or percutaneous interventions. However, these interventions cannot be performed in a significant portion of patients, and many do not respond to these therapies. An alternative approach for treating PAD is to use proteins to stimulate the body to create new vasculature, thus restoring blood flow through its own regenerative processes. Stem cell factor (SCF) is a cytokine that acts through the receptor tyrosine kinase c-Kit to regulate hematopoiesis and has been a candidate protein for treating PAD. Clinical use of soluble SCF would be highly beneficial but has been limited due to toxicity related to mast cell activation. SCF also exists in a transmembrane form that has differential activity from soluble SCF and has not been explored as a therapeutic agent. To explore the transmembrane SCF (tmSCF) as a therapeutic we created formulations of tmSCF embedded in proteoliposomes or in lipid nanodiscs. Mouse models of anaphylaxis and ischemia revealed the tmSCF-based therapies did not activate mast cells and were effective in improving the recovery from ischemia in both wild type and diabetic mice. We also found that the formulation of the lipid nanocarrier to deliver tmSCF altered the biological response and trophism of the tmSCF-based treatments. Proteoliposomal tmSCF preferentially acted on mature endothelial cells to induce angiogenesis while tmSCF nanodiscs had greater activity in inducing stem cell mobilization from the bone marrow and recruitment to ischemic sites. A mechanistic analysis of the effects of the treatments on mast cells, mature endothelial cells and endothelial progenitor cells, revealed that the nanocarriers altered the relative utilization of clathrin- versus caveolin-mediated uptake pathways of c-Kit in response to the treatments. Overall, our studies support tmSCF-based therapies can provide therapeutic benefits without off-target effects on mast cells and that lipid nanocarriers can be used to tailor the properties of membrane protein-based therapeutics.
Widespread Misinterpretable ChIP-seq Bias in Yeast
(PLOS One, 2013-12-09) Park, Daechan; Lee, Yaelim; Bhupindersingh, Gurvani; Lyer, Vishwanath R.
Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is widely used to detect genome-wide interactions between a protein of interest and DNA in vivo. Loci showing strong enrichment over adjacent background regions are typically considered to be sites of binding. Insufficient attention has been given to systematic artifacts inherent to the ChIP-seq procedure that might generate a misleading picture of protein binding to certain loci. We show here that unrelated transcription factors appear to consistently bind to the gene bodies of highly transcribed genes in yeast. Strikingly, several types of negative control experiments, including a protein that is not expected to bind chromatin, also showed similar patterns of strong binding within gene bodies. These false positive signals were evident across sequencing platforms and immunoprecipitation protocols, as well as in previously published datasets from other labs. We show that these false positive signals derive from high rates of transcription, and are inherent to the ChIP procedure, although they are exacerbated by sequencing library construction procedures. This expression bias is strong enough that a known transcriptional repressor like Tup1 can erroneously appear to be an activator. Another type of background bias stems from the inherent nucleosomal structure of chromatin, and can potentially make it seem like certain factors bind nucleosomes even when they don't. Our analysis suggests that a mock ChIP sample offers a better normalization control for the expression bias, whereas the ChIP input is more appropriate for the nucleosomal periodicity bias. While these controls alleviate the effect of the biases to some extent, they are unable to eliminate it completely. Caution is therefore warranted regarding the interpretation of data that seemingly show the association of various transcription and chromatin factors with highly transcribed genes in yeast.