Investigating the HIV-1 Tat-TAR interaction
The HIV-1 Tat protein is integral to the viral life-cycle as it can induce efficient transcription of the virus after binding a folded element of the HIV LTR called TAR. Previous studies have elucidated the effects of some mutations of Tat, but the overall depth and density of the studied mutations is low. We are investigating the Tat-TAR interaction using deep mutational scanning, a high-throughput technology recently developed in the lab.
By creating a library of hundreds of thousands of variants of Tat and selecting for binding to TAR using a yeast three-hybrid assay, we are examining the relationship between the sequence of Tat and its TAR-binding function at an unprecedented resolution. The Tat-TAR interaction is thought to be driven by an enrichment of basic residues in the core of the protein rather than a specific amino acid sequence, but it is not known if point mutations outside of this core region can affect the TAR interaction. This study of mutations that affect the affinity of Tat to TAR can contribute to our understanding of both protein-RNA interactions, as well as the mechanism of HIV transcription and activation.
•Daniel Melamed & Matt Rich
Synonymous Variation and Fitness in a Yeast Model Gene
Although synonymous codons encode identical amino acids, variation in a gene’s synonymous codons can lead to subtle alterations in protein production and exert significant phenotypic effects. To investigate factors important to synonymous codon usage and protein production, we are using yeast HIS3 as a model gene. HIS3 encodes an enzyme required for the synthesis of histidine. After constructing four plasmid libraries of synonymous HIS3 variants, we will compete cells carrying the variants for their ability to grow in media lacking histidine. Cells in a population that carry HIS3 variants with beneficial synonymous changes increase in the population based upon the competitive fitness of their HIS3 genes, whereas cells carrying deleterious variants decrease. After recovering the DNA plasmids from population samples grown under histidine selection, we use high-throughput DNA sequencing to measure the relative abundance of each variant and to calculate variant enrichment scores. With these data, we can begin to explore the relative fitness contributions of factors such as mRNA secondary structure and codon usage bias.
Genome-wide analysis of nascent transcription in Saccharomyces cerevisiae
Most studies of eukaryotic gene regulation have examined mature, steady-state mRNA levels. However, steady-state mRNA levels result from the action of two opposing processes: RNA synthesis and RNA degradation. An accurate assessment of RNA synthesis is important for understanding the mechanisms that regulate gene expression.
The nuclear run-on (NRO) assay is the traditional method to directly measure RNA synthesis. We have combined the in vivo RNA labeling of this assay with high throughput DNA sequencing to examine RNA polymerase activity genome-wide in exponentially growing yeast. In parallel, we sequenced total RNA to monitor transcript abundance and compare nascent transcript and steady-state transcript levels (Figure 1A).
To analyze RNA polymerase activity within genes, we examined read density along transcribed regions. We find that in contrast to total RNA libraries, NRO libraries show a high density of reads near the 5’ ends of the transcript models, with a peak ~50 bp downstream of the transcription start site (TSS) (Figure 1B), as has been observed in human and Drosophila cells. This peak in read depth near TSSs likely indicates a promoter-proximal accumulation of paused RNA polymerase, suggesting that pausing plays a significant role in the regulation of yeast transcription. Analysis of expression levels allows us to classify genes into four classes by their activity and pausing (Figure 1C). Ranking genes by the significance of pausing reveals that histone genes are among the 5% most paused genes, suggesting that transition to productive elongation is necessary for rapid induction of histone synthesis in S phase. By calculating the ratio of NRO transcription to total RNA for each gene, we can estimate nascent transcript stabilities. This analysis has revealed that the most stable and unstable transcripts encode proteins whose functional roles are consistent with these stabilities.
Parallel analysis of nascent transcripts and steady-state transcripts with high throughput sequencing allows a genome-wide assessment of RNA polymerase activity in yeast, identifying regulatory steps of RNA synthesis and inference of RNA stabilities. We anticipate that this approach will be useful to measure changes that occur in transcription in response to environmental or genetic perturbations.
Published Results:
McKinlay, A., Araya, C.L. and Fields, S. Genome-wide analysis of nascent transcription in Saccharomyces cerevisiae. G3: Genes, Genomes, Genetics, 2011. in press.
•Anastasia McKinlay & Carlos Araya (former lab members)
Capture and sequence analysis of RNAs containing 3' cyclic phosphate termini
Standard techniques used to isolate and identify RNA from cellular extracts have traditionally relied upon hybridization to oligo-dT or T4 RNA ligase-based methodologies. These methods have been successful in isolating populations of RNAs that are modified with poly-adenosine tracts or have hydroxyl moieties (-OH) at their 3’ terminus. It is possible that these two classes represent the majority of the cellular ‘RNA universe.’ However, with the development of advanced sequencing technologies, it is also clear that the RNA universe is more complex than previously appreciated. Therefore, there is a need to develop new technologies to further profile this complexity.
With this in mind we developed a technology that is capable of specifically isolating 2’,3’ cyclic phosphate-terminated RNAs from complex RNA mixtures. RNAs with these termini are generated as the product of particular RNA endonucleases or during ribonucleolytic cleavage. This technology uses the Arabidopsis thaliana tRNA ligase to add an adaptor oligonucleotide to RNAs that terminate in 2’,3’ cyclic phosphates. The adaptor allows specific priming by reverse transcriptase, which is followed by additional steps for PCR amplification and high throughput DNA sequencing. This method may identify processing events previously undetected by other RNA cloning techniques.
|
       |
|
|
