VI. Validated new techniques for hybridization capture analysis of RNA- and DNA-binding proteins

Revealing protein interactomes for DNA loci and RNAs

Ensembles of proteins bind RNA and DNA and have typically been profiled by immunoprecipitation (IP) methods that pull down proteins along with their nucleic acid counterparts, which can then be processed by nucleic acid sequencing. However, the full ensemble of proteins located at a specific site may vary between conditions, and this technique also can miss posttranslational modifications (PTMs) that may be present or specific to DNA or RNA loci. I took part in an effort to create and validate a hybridization capture assay that targets nucleic acids rather than protein and uses mass spectrometry to analyze the proteins, including possible PTMs. Specifically, I contributed to this work by validating the results notably by using existing IP-based sequencing datasets. These studies provide a look at the RNA-binding proteins of known oncogenes, DNA loci in yeast, and long noncoding RNAs (lncRNAs) enriched in certain human cancers. These techniques that I helped validate have been applied in follow-up studies to various spliced HIV-1 RNAs to reveal differences in RNA-binding complements of these important splice forms.

Research papers

(1) Spiniello, M.; Knoener, R. A.; Steinbrink, M. I.; Yang, B.; Cesnik, A. J.; Buxton, K. E.; Scalf, M.; Jarrard, D. F.; Smith, L. M. “HyPR­-MS for multiplexed discovery of the MALAT1, NEAT1, and NORAD lncRNA protein interactomes.” Journal of Proteome Research 2018, 17, 9, 3022–3038.

(2) Spiniello, M.; Steinbrink, M. I.; Cesnik, A. J.; Miller, R. M.; Scalf, M.; Shortreed, M. R.; Smith, L. M. “Comprehensive in vivo identification of the c­-Myc mRNA protein interactome using HyPR­-MS.” RNA 2019, 25, 1337–1352.

(3) Guillen-­Ahlers, H.; Rao, P. K.; Levenstein, M. E.; Kennedy-­Darling, J.; Perumalla, D. S.; Jadhav, A. Y. L.; Glenn, J. P.; Ludwig-­Kubinski, A.; Drigalenko, E.; Montoya, M. J.; Göring, H. H.; Anderson, C. D.; Scalf, M.; Gildersleeve, H. I.S.; Cole, R.; Greene, A. M. Oduro, A. K.; Lazarova, K.; Cesnik, A. J.; Barfknecht, J.; Cirillo, L. A.; Gasch, A. P.; Shortreed, M. R.; Smith, L. M.; Olivier, M. “HyCCAPP as a tool to characterize promoter DNA-­protein interactions in Saccharomyces cerevisiae.” Genomics 2016, 107, 6, 267–273.