Genome-wide mRNA expression correlates of viral control in CD4+ T-cells from HIV-1-infected individuals.

There is great interindividual variability in HIV-1 viral setpoint after seroconversion, some of which is known to be due to genetic differences among infected individuals. Here, our focus is on determining, genome-wide, the contribution of variable gene expression to viral control, and to relate it to genomic DNA polymorphism. RNA was extracted from purified CD4+ T-cells from 137 HIV-1 seroconverters, 16 elite controllers, and 3 healthy blood donors. Expression levels of more than 48,000 mRNA transcripts were assessed by the Human-6 v3 Expression BeadChips (Illumina). Genome-wide SNP data was generated from genomic DNA using the HumanHap550 Genotyping BeadChip (Illumina). We observed two distinct profiles with 260 genes differentially expressed depending on HIV-1 viral load. There was significant upregulation of expression of interferon stimulated genes with increasing viral load, including genes of the intrinsic antiretroviral defense. Upon successful antiretroviral treatment, the transcriptome profile of previously viremic individuals reverted to a pattern comparable to that of elite controllers and of uninfected individuals. Genome-wide evaluation of cis-acting SNPs identified genetic variants modulating expression of 190 genes. Those were compared to the genes whose expression was found associated with viral load: expression of one interferon stimulated gene, OAS1, was found to be regulated by a SNP (rs3177979, p = 4.9E-12); however, we could not detect an independent association of the SNP with viral setpoint. Thus, this study represents an attempt to integrate genome-wide SNP signals with genome-wide expression profiles in the search for biological correlates of HIV-1 control. It underscores the paradox of the association between increasing levels of viral load and greater expression of antiviral defense pathways. It also shows that elite controllers do not have a fully distinctive mRNA expression pattern in CD4+ T cells. Overall, changes in global RNA expression reflect responses to viral replication rather than a mechanism that might explain viral control.

The Role of mRNA Translation in the Regulation of Ribosome Trafficking to the Endoplasmic Reticulum

The goal of this research is to identify the trafficking patterns that direct ribosomes to the endoplasmic reticulum (ER). It is widely believed that the SRP pathway is the only mechanism that cells use to localize mRNA and ribosomes to the ER, but this has been found not to be a sufficient explanation for the patterns of RNA localization in cells, namely that non-signal sequence-containing mRNA are translated on the ER and that ribosomes retain their membrane association after translation termination. First, a summary of the history of the field is presented to provide context for the key, unanswered questions in the field. Then, experiments employing [32Pi] pulse-chase labeling of HeLa cells over a time course to follow nascent ribosome trafficking are presented. The purpose of the cell labeling was to track rRNA processing and assembly into nascent ribosomes, followed by their export into the cytoplasm and recruitment into active polysomes. A detergent-based cell fractionation procedure was also utilized to separate the cytosol and ER compartments in order to observe ribosomes on their path as they exit the nucleus and either localize to the ER or cytosolic cellular compartment. Through this method, it was seen that ribosomes appear in both compartments at the same time, suggesting a mechanism may be occurring in addition to SRP-dependent ribosome trafficking. This research provides an understanding toward a mechanism that is not currently known, but will one day more fully explain the patterns of ribosomal localization.

Nutritional control of mRNA isoform expression during developmental arrest and recovery in C. elegans.

Nutrient availability profoundly influences gene expression. Many animal genes encode multiple transcript isoforms, yet the effect of nutrient availability on transcript isoform expression has not been studied in genome-wide fashion. When Caenorhabditis elegans larvae hatch without food, they arrest development in the first larval stage (L1 arrest). Starved larvae can survive L1 arrest for weeks, but growth and post-embryonic development are rapidly initiated in response to feeding. We used RNA-seq to characterize the transcriptome during L1 arrest and over time after feeding. Twenty-seven percent of detectable protein-coding genes were differentially expressed during recovery from L1 arrest, with the majority of changes initiating within the first hour, demonstrating widespread, acute effects of nutrient availability on gene expression. We used two independent approaches to track expression of individual exons and mRNA isoforms, and we connected changes in expression to functional consequences by mining a variety of databases. These two approaches identified an overlapping set of genes with alternative isoform expression, and they converged on common functional patterns. Genes affecting mRNA splicing and translation are regulated by alternative isoform expression, revealing post-transcriptional consequences of nutrient availability on gene regulation. We also found that phosphorylation sites are often alternatively expressed, revealing a common mode by which alternative isoform expression modifies protein function and signal transduction. Our results detail rich changes in C. elegans gene expression as larvae initiate growth and post-embryonic development, and they provide an excellent resource for ongoing investigation of transcriptional regulation and developmental physiology.

Sensitive and precise quantification of insulin-like mRNA expression in Caenorhabditis elegans.

Insulin-like signaling regulates developmental arrest, stress resistance and lifespan in the nematode Caenorhabditis elegans. However, the genome encodes 40 insulin-like peptides, and the regulation and function of individual peptides is largely uncharacterized. We used the nCounter platform to measure mRNA expression of all 40 insulin-like peptides as well as the insulin-like receptor daf-2, its transcriptional effector daf-16, and the daf-16 target gene sod-3. We validated the platform using 53 RNA samples previously characterized by high density oligonucleotide microarray analysis. For this set of genes and the standard nCounter protocol, sensitivity and precision were comparable between the two platforms. We optimized conditions of the nCounter assay by varying the mass of total RNA used for hybridization, thereby increasing sensitivity up to 50-fold and reducing the median coefficient of variation as much as 4-fold. We used deletion mutants to demonstrate specificity of the assay, and we used optimized conditions to assay insulin-like gene expression throughout the C. elegans life cycle. We detected expression for nearly all insulin-like genes and find that they are expressed in a variety of distinct patterns suggesting complexity of regulation and specificity of function. We identified insulin-like genes that are specifically expressed during developmental arrest, larval development, adulthood and embryogenesis. These results demonstrate that the nCounter platform provides a powerful approach to analyzing insulin-like gene expression dynamics, and they suggest hypotheses about the function of individual insulin-like genes.

The DLK-1 kinase promotes mRNA stability and local translation in C. elegans synapses and axon regeneration.

Growth cone guidance and synaptic plasticity involve dynamic local changes in proteins at axons and dendrites. The Dual-Leucine zipper Kinase MAPKKK (DLK) has been previously implicated in synaptogenesis and axon outgrowth in C. elegans and other animals. Here we show that in C. elegans DLK-1 regulates not only proper synapse formation and axon morphology but also axon regeneration by influencing mRNA stability. DLK-1 kinase signals via a MAPKAP kinase, MAK-2, to stabilize the mRNA encoding CEBP-1, a bZip protein related to CCAAT/enhancer-binding proteins, via its 3'UTR. Inappropriate upregulation of cebp-1 in adult neurons disrupts synapses and axon morphology. CEBP-1 and the DLK-1 pathway are essential for axon regeneration after laser axotomy in adult neurons, and axotomy induces translation of CEBP-1 in axons. Our findings identify the DLK-1 pathway as a regulator of mRNA stability in synapse formation and maintenance and also in adult axon regeneration.