Browsing by Subject "Post-transcriptional"

N6-methyladenosine (m6A) is deposited co-transcriptionally on thousands of cellular mRNAs and plays important roles in mRNA processing and cellular function. m6A is particularly abundant within the brain and is critical for neurodevelopment. However, the mechanisms through which m6A contributes to brain development are inco¬¬mpletely understood. Here, we discover serine-/arginine-rich splicing factor 7 (SRSF7) and RNA-binding motif-containing protein 45 (RBM45) as m6A-binding proteins in transformed hippocampal neurons. We find that SRSF7 binds to exon-intron junctions in methylated pre-mRNA targets and regulates the gene expression of thousands of cellular mRNAs, including the m6A RNA methyltransferase, METTL3. We find that RBM45 binds to thousands of cellular RNAs, predominantly within intronic regions. Rbm45 depletion disrupts the constitutive splicing of a subset of target pre-mRNAs, leading to altered mRNA and protein levels through both m6A-dependent and m6A-independent mechanisms. Finally, we find that RBM45 is highly expressed during embryonic neurodevelopment, demonstrating that expression of RBM45 is necessary for neuroblastoma cell differentiation and that its depletion impacts the expression of genes involved in several neurodevelopmental signaling pathways. Altogether, our findings identify roles for SRSF7 and RBM45 in gene expression regulation, and highlight a previously unknown function for RBM45 in the control of pre-mRNA processing and neuronal differentiation, mediated in part by the recognition of methylated RNA.

CD8+ T cells are associated with long term control of virus replication to low or undetectable levels in a population of HIV+ therapy-naïve individuals known as virus controllers (VCs; <5000 RNA copies/ml and CD4+ lymphocyte counts >400 cells/µl). These subjects' ability to control viremia in the absence of therapy makes them the gold standard for the type of CD8+ T-cell response that should be induced with a vaccine. Studying the regulation of CD8+ T cells responses in these VCs provides the opportunity to discover mechanisms of durable control of HIV-1. Previous research has shown that the CD8+ T cell population in VCs is heterogeneous in its ability to inhibit virus replication and distinct T cells are responsible for virus inhibition. Further defining both the functional properties and regulation of the specific features of the select CD8+ T cells responsible for potent control of viremia the in VCs would enable better evaluation of T cell-directed vaccine strategies and may inform the design of new therapies.

Here we discuss the progress made in elucidating the features and regulation of CD8+ T cell response in virus controllers. We first detail the development of assays to quantify CD8+ T cells' ability to inhibit virus replication. This includes the use of a multi-clade HIV-1 panel which can subsequently be used as a tool for evaluation of T cell directed vaccines. We used these assays to evaluate the CD8+ response among cohorts of HIV-1 seronegative, HIV-1 acutely infected, and HIV-1 chronically infected (both VC and chronic viremic) patients. Contact and soluble CD8+ T cell virus inhibition assays (VIAs) are able to distinguish these patient groups based on the presence and magnitude of the responses. When employed in conjunction with peptide stimulation, the soluble assay reveals peptide stimulation induces CD8+ T cell responses with a prevalence of Gag p24 and Nef specificity among the virus controllers tested. Given this prevalence, we aimed to determine the gene expression profile of Gag p24-, Nef-, and unstimulated CD8+ T cells. RNA was isolated from CD8+ T-cells from two virus controllers with strong virus inhibition and one seronegative donor after a 5.5 hour stimulation period then analyzed using the Illumina Human BeadChip platform (Duke Center for Human Genome Variation). Analysis revealed that 565 (242 Nef and 323 Gag) genes were differentially expressed in CD8+ T-cells that were able to inhibit virus replication compared to those that could not. We compared the differentially expressed genes to published data sets from other CD8+ T-cell effector function experiments focusing our analysis on the most recurring genes with immunological, gene regulatory, apoptotic or unknown functions. The most commonly identified gene in these studies was TNFRSF9. Using PCR in a larger cohort of virus controllers we confirmed the up-regulation of TNFRSF9 in Gag p24 and Nef-specific CD8+ T cell mediated virus inhibition. We also observed increase in the mRNA encoding antiviral cytokines macrophage inflammatory proteins (MIP-1α, MIP-1αP, MIP-1β), interferon gamma (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), and recently identified lymphotactin (XCL1).

Our previous work suggests the CD8+ T-cell response to HIV-1 can be regulated at the level of gene regulation. Because RNA abundance is modulated by transcription of new mRNAs and decay of new and existing RNA we aimed to evaluate the net rate of transcription and mRNA decay for the cytokines we identified as differentially regulated. To estimate rate of mRNA synthesis and decay, we stimulated isolated CD8+ T-cells with Gag p24 and Nef peptides adding 4-thiouridine (4SU) during the final hour of stimulation, allowing for separation of RNA made during the final hour of stimulation. Subsequent PCR of RNA isolated from these cells, allowed us to determine how much mRNA was made for our genes of interest during the final hour which we used to calculate rate of transcription. To assess if stimulation caused a change in RNA stability, we calculated the decay rates of these mRNA over time. In Gag p24 and Nef stimulated T cells , the abundance of the mRNA of many of the cytokines examined was dependent on changes in both transcription and mRNA decay with evidence for potential differences in the regulation of mRNA between Nef and Gag specific CD8+ T cells. The results were highly reproducible in that in one subject that was measured in three independent experiments the results were concordant.

This data suggests that mRNA stability, in addition to transcription, is key in regulating the direct anti-HIV-1 function of antigen-specific memory CD8+ T cells by enabling rapid recall of anti-HIV-1 effector functions, namely the production and increased stability of antiviral cytokines. We have started to uncover the mechanisms employed by CD8+ T cell subsets with antigen-specific anti-HIV-1 activity, in turn, enhancing our ability to inhibit virus replication by informing both cure strategies and HIV-1 vaccine designs that aim to reduce transmission and can aid in blocking HIV-1 acquisition.

Precise regulation of the complex process of gene expression is essential for all aspects of life, and a large degree of this precision is mediated at the posttranscriptional level. The global and individual mechanisms by which posttranscriptional control is coordinated to maintain or alter levels of gene expression as necessary are not fully understood. Identification of the mRNA target sets of individual RNA binding proteins (RBPs) and characterization of the mechanisms by which RBPs regulate the expression of individual mRNAs provide some insight into the global structure of the posttranscriptional environment. However, few studies have integrated these findings into a global model of posttranscriptional control. We have explored the structure and function of the posttranscriptional regulatory system through a combination of global modeling approaches, global studies of mRNA translation and decay, and mechanistic studies of the function of individual RBPs, specifically HuR and Pum1.

By combining RBP-mRNA association data and transcription factor (TF) target data from separate global studies in yeast, we developed an integrated model of gene expression regulation. Evaluation of this model indicates that posttranscriptional regulation may be responsible for substantially greater contributions to the overall gene expression program than transcriptional regulation. Further, we identified a self-regulatory feature of the posttranscriptional network that suggests a `regulators of regulators' structure may be a defining feature of the posttranscriptional control of gene expression.

Additionally, we explored the mechanisms and functional consequences of the dynamic association between an RBP, HuR, and its target RNAs through a combination of modeling and experimental approaches, including polysome profile analysis and global measurement of RNA stability. Our model indicates that changes in total mRNA abundance are insufficient to fully explain the dynamics of association between HuR and its targets, suggesting a role for competition and cooperation with other RBPs. Our findings also indicate that HuR may play a role in inhibition of translation in a dynamic immunological system (T cell activation).

Finally, we performed a mechanistic analysis of the function of the Pum1 RBP and characterized the role of this protein in the translational regulation of several important target mRNAs through the use of luciferase reporter assays. We also provided the first in vivo evidence of a role for specific regions of the Pum1 protein in the mediation of gene expression. However, we were unable to verify previous in vitro reports of a role for Pum1 in the control of translation elongation of verified in vivo mRNA targets, suggesting that Pum1's regulatory function may be context dependent.

Ultimately, the approaches and findings in this study will provide a framework for the development of a global integrated model of posttranscriptional control. Through the iterative development of models and experimentation, hypotheses can be generated and, tested in the laboratory, and the results of these experiments will then further improve the development of the models. An integrated approach of this type will be necessary to fully understand the highly complex and interconnected nature of the gene expression regulatory system.