RNA-protein-interactions

Technology: HyPR-MS is a strategy developed by our lab that for the selective isolation and identification of individual RNA-protein interactomes. In HyPR-MS, cells are formaldehyde crosslinked to stabilize RNA-protein interactions, followed by cell lysis and sequence-specific RNA hybridization to capture and purify the target RNA molecules and their associated proteins. The protein interactors are then identified via mass spectrometry. HyPR-MS is capable of being multiplexed and is effective for various classes of RNA molecules, making it both a powerful and effective technique.

Significance: RNA-protein interactions are vital to many biological processes such as transcription, nuclear organization, splicing and translation [1]. Some proteins associate with RNA in a site- specific manner while others are more promiscuous in their binding. Dysregulation of RNA-Protein interactions plays a crucial role in certain disease states. Therefore, knowledge of the RNA-binding proteome is essential to understanding RNA biology and developing efficient drug therapies [2]. Many different technologies exist to elucidate these RNA-protein interactions. Both eClip (enhanced crosslinking and immunoprecipitation) [3] and RIP-ChIP (RNA immunoprecipitation ChIP) [4] are protein-centric approaches that are widely used to determine what RNAs select proteins associate with. A limitation of these protein centric approaches is that they do not provide any additional information about other proteins that are bound to the same RNA as the bait protein.

Figure 1. HIV RNA-Protein interactor map generated using HyPR-MS. Figure from [5].

HyPR-MS [5], ChiRP-MS (comprehensive identification of RNA-binding proteins by mass spectrometry) [6], CHART-MS (capture hybridization analysis of RNA targets and mass spectrometry) [7] and RAP-MS (RNA antisense purification and mass spectrometry) [8] are RNA-centric approaches. These approaches allow for many protein interactors for a single RNA to be elucidated at once. The success of RNA-centric approaches is dependent on the design of the capture oligonucleotides. The unique toehold design of the probes used for HyPR-MS ameliorates some of the major design limitations that plague other RNA-centric approaches. Additionally, RNA-Interaction studies can be costly and labor-intensive. HyPR-MS is unique in its ability to be multiplexed, meaning that from one aliquot of cell lysate, multiple capture probes for different RNAs can be used to determine their protein interactors. Each RNA and its associated proteins are eluted sequentially for independent mass spectrometry analysis making each experiment more efficient and cost effective [9].

Figure 2. Multiplexed hybridization purification of RNA-protein complexes for MS analysis. Figure from [9]

We have several current, large-scale projects and collaborations in our lab utilizing HyPR-MS. We have also identified some exciting new future directions for this technology.

HyPR-MS Publications:

Knoener, R. A.; Becker, J. T.; Scalf, M.; Sherer, N. M.; Smith, L. M. Elucidating the in vivo interactome of HIV-1 RNA by hybridization capture and mass spectrometry. Scientific Reports. 2017, 7: 16965

Spiniello, M.; Knoener, R. A.; Steinbrink, M. I.; Yang, B.; Cesnik, A.; Buxton, K. E.; Scalf, M.; Jarrard, D. F.; Smith, L. M. HyPR-MS for Multiplexed Discovery of MALAT1, NEAT1 and NORAD lncRNA Protein Interactomes. J.Proteome Res. 2018, 17(9), 3022-3038.

Spiniello, M.; Knoener, R. A.; Steinbrink, M. I.; Cesnik, A.; Miller, R. M.; Scalf, M.; Shortreed, M. R.; Smith, L. M. Comprehensive in vivo identification of the c-Myc mRNA interactome using HyPR-MS. RNA. 2019

References:

1. Khalil, A. M.; Rinn, J. L. RNA-protein interactions in human health and disease. Semin. Cell Dev. Biol. 2011, 22 (4), 359– 65
2. Marchese, D.; de Groot, N. S.; Lorenzo Gotor, N.; Livi, C. M.; Tartaglia, G. G. Advances in the characterization of RNA-binding proteins. Wiley Interdiscip Rev. RNA 2016, 7 (6), 793– 810
3. Van Nostrand, et. al, Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP), Nature Methods (2016),13: 508–514
4. Keene, J. D.; Komisarow, J. M.; Friedersdorf, M. B. RIP-Chip: the isolation and identification of mRNAs, microRNAs and protein components of ribonucleoprotein complexes from cell extracts. Nat. Protoc. 2006. 1(1), 302-7.
5. Knoener, R. A.; Becker, J. T.; Scalf, M.; Sherer, N. M.; Smith, L. M. Elucidating the in vivo interactome of HIV-1 RNA by hybridization capture and mass spectrometry. Scientific Reports. 2017, 7: 16965
6. Chu, C.; Zhang, Q. C.; da Rocha, S. T.; Flynn, R. A.; Bharadwaj, M.; Calabrese, J. M.; Magnuson, T.; Heard, E.; Chang, H. Y. Systematic discovery of Xist RNA binding proteins. Cell 2015, 161 (2), 404– 16
7. West, J. A.; Davis, C. P.; Sunwoo, H.; Simon, M. D.; Sadreyev, R. I.; Wang, P. I.; Tolstorukov, M. Y.; Kingston, R. E. The long noncoding RNAs NEAT1 and MALAT1 bind active chromatin sites. Mol. Cell 2014, 55 (5), 791– 802
8. McHugh, C. A.; Chen, C. K.; Chow, A.; Surka, C. F.; Tran, C.; McDonel, P.; Pandya-Jones, A.; Blanco, M.; Burghard, C.; Moradian, A.; Sweredoski, M. J.; Shishkin, A. A.; Su, J.; Lander, E. S.; Hess, S.; Plath, K.; Guttman, M. The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3. Nature 2015, 521 (7551), 232– 6
9. Spiniello, M.; Knoener, R. A.; Steinbrink, M. I.; Yang, B.; Cesnik, A.; Buxton, K. E.; Scalf, M.; Jarrard, D. F.; Smith, L. M. HyPR-MS for Multiplexed Discovery of MALAT1, NEAT1 and NORAD lncRNA Protein Interactomes. J.Proteome Res. 2018, 17(9), 3022-3038.