Overview: RNA molecules are associated with proteins in complexes for their entire lifecycle. These complexes are subject to dynamic remodeling, nuclear export, localization, storage, translation, and degradation. Alteration of the expression, sequence, post-translational modifications, or binding cofactors of either a protein or its RNA regulatory sequence can disrupt normal interactions that have been linked to neurological, metabolic, and tumoral diseases. In-depth characterization of specific RNA-bound proteomes is essential to understanding RNA biology in physiological and pathological conditions and to identifying new therapeutic targets.

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.

Current Projects:

• Adapt HyPR-MS protocol for the elucidation of Malat-1 RNA-proteoform interactions - (Graduate students Katie Buxton and Rachel Miller)
  
  • Up to this point all of the HyPR-MS studies have utilized bottom-up proteomics to determine which proteins are associated with the target RNA. This means that once the target RNA and its associated proteins are captured, proteases are used to digest the proteins into peptides which are then identified via mass spectrometry. Bottom-up proteomics, although robust, cannot be used to identify proteoforms. A proteoform is a defined form of a protein derived from a given gene with a specific amino acid sequence and localized post-translational modification. The biological function of different proteoforms can vary greatly, even among those derived from the same gene. This biological importance is why it is important to identify and characterize the proteoforms associated with a RNA target, different RNA proteoforms might be selectively associated with various disease states.
• Use HyPR-MS to determine differential protein interactors for the various splice isoforms of HIV RNA -(Post-Doc Rachel Knoener in collaboration with Professor Nathan Sherer)
  
  • The HIV-1 primary transcript undergoes extensive alternative splicing resulting in over 50 mRNA isoforms. These isoforms fall into three classes; unspliced, partially spliced or completely spliced, and serve various functions in the viral life cycle. HyPR-MS is utilized to determine the protein interactors of the three classes of isoforms to elucidate host dependency and restriction factors affecting the various stages of the viral life cycle.
• Use HyPR-MS to study the protein interactome of hepatitis B virus (HBV) - (Post-Doc Rachel Knoener in collaboration with graduate student Sofia Ferrufino and Professor Nathan Sherer)
  
  • Hepatitis B virus is one of the few known non-retroviruses that still uses reverse transcription in their viral replication process. The genome of HBV is circular and, at times, not fully double-stranded. HyPR-MS will be used to target the viral RNA as well as the single stranded region of the HBV DNA genome in order to determine which host proteins the viral genome interacts with and gain further insight into the pathogenesis of this highly infectious disease.
• Use HyPR-MS to differentiate the host factors necessary for viral RNA nuclear export among lentiviruses and deltaretroviruses - (Post-Doc Rachel Knoener in collaboration with Post-Doc Ryan Behrens and Professor Nathan Sherer)
  
  • The lentivirus and deltaretrovirus members of the HIV-1 evolutionary tree share functionally conserved protein and RNA components necessary for viral RNA nuclear export. Though these components share some functionality, their subcellular localization patterns can be quite different. We will use HyPR-MS to determine the shared and/or different cellular proteins that play a role in these nuclear export pathways through association with the viral RNAs.

Future Projects:

• Currently, our HyPR-MS technology is limited to relatively high abundance transcripts. Increasing the efficiency of the both RNA capture and release would allow for the application of HyPR-MS to lower abundance transcripts. This is important because many interesting, and disease related transcripts are low abundance.
• All HyPR-MS experiments up to this point have used cultured cells. The adaptation of HyPR-MS to function utilizing tissue samples would open the door to various studies using mice or human donor that could better mimic clinical applications.

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.