rec-Y3H screening allows the detection of simultaneous RNA-protein interface mutations

Garriga-Canut, Mireia; Yang, Jun; Preußer, Friedrich; Speroni, Silvia; Gili, Maria; Maurer, Sebastian P.

doi:10.1016/j.ymeth.2019.09.002

Cited by 5 publications

(3 citation statements)

References 20 publications

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“…Discovering essential building blocks of the neuronal mRNA transport machinery: while pulldown studies have provided a valuable pool of proteins that are involved in mRNA transport, they might fail to capture important transient interactions and do not report direct interactions. As a complementary technique, high-throughput (HT) screening for direct interactions with proper orthogonal validations could add valuable information to understand how mRNAs, mRNA-motor adaptors, and motor proteins directly interact (Yang et al, 2018 ; Garriga-Canut et al, 2019 ; Lang et al, 2020 ). HT screening can also help to narrow down essential linkers by interaction network analysis and function as a hypothesis-generator for subsequent mechanistic studies.…”

Section: Perspectivesmentioning

confidence: 99%

Mammalian Neuronal mRNA Transport Complexes: The Few Knowns and the Many Unknowns

Rodrigues

Grawenhoff

Baumann

et al. 2021

Front. Integr. Neurosci.

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Hundreds of messenger RNAs (mRNAs) are transported into neurites to provide templates for the assembly of local protein networks. These networks enable a neuron to configure different cellular domains for specialized functions. According to current evidence, mRNAs are mostly transported in rather small packages of one to three copies, rarely containing different transcripts. This opens up fascinating logistic problems: how are hundreds of different mRNA cargoes sorted into distinct packages and how are they coupled to and released from motor proteins to produce the observed mRNA distributions? Are all mRNAs transported by the same transport machinery, or are there different adaptors or motors for different transcripts or classes of mRNAs? A variety of often indirect evidence exists for the involvement of proteins in mRNA localization, but relatively little is known about the essential activities required for the actual transport process. Here, we summarize the different types of available evidence for interactions that connect mammalian mRNAs to motor proteins to highlight at which point further research is needed to uncover critical missing links. We further argue that a combination of discovery approaches reporting direct interactions, in vitro reconstitution, and fast perturbations in cells is an ideal future strategy to unravel essential interactions and specific functions of proteins in mRNA transport processes.

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Section: Perspectivesmentioning

confidence: 99%

Mammalian Neuronal mRNA Transport Complexes: The Few Knowns and the Many Unknowns

Rodrigues

Grawenhoff

Baumann

et al. 2021

Front. Integr. Neurosci.

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“…Moreover, while some of these networks do have considerable RBP bait representation, they fail to assess the RNA dependency of these interactions, and therefore cannot distinguish if interactions between two proteins are direct or RNA-mediated. RBP-focused attempts to delineate RBP PPI networks also have similar limitations, including lack of RNA-aware interaction information and use of exogenous tags to immunoprecipitate or localize the RBPs (Garriga-Canut et al 2020;Kwon et al 2013). For example, the yeast two-hybrid screen (rec-Y2H) (Lang et al 2021) generated RNA-independent pairwise binding maps of 978 human RBPs, however the incorporation of nuclear localization signals in each protein likely resulted in some spurious interactions.…”

Section: Introductionmentioning

confidence: 99%

Large-scale map of RNA binding protein interactomes across the mRNA life-cycle

Street,

Rothamel,

Brannan

et al. 2023

Preprint

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Messenger RNAs interact with RNA-binding proteins (RBPs) in diverse ribonucleoprotein complexes (RNPs) during distinct life-cycle stages for their processing and maturation. While substantial attention has focused on understanding RNA regulation by assigning proteins, particularly RBPs, to specific RNA substrates, there has been considerably less exploration leveraging protein-protein interaction (PPI) methodologies to identify and study the role of proteins in mRNA life-cycle stages. To address this gap, we generated an RNA-aware RBP-centric PPI map across the mRNA life-cycle by immunopurification (IP-MS) of ~100 endogenous RBPs across the life-cycle in the presence or absence of RNase, augmented by size exclusion chromatography (SEC-MS). Aside from confirming 8,700 known and discovering 20,359 novel interactions between 1125 proteins, we determined that 73% of our IP interactions are regulated by the presence of RNA. Our PPI data enables us to link proteins to life-cycle stage functions, highlighting that nearly half of the proteins participate in at least two distinct stages. We show that one of the most highly interconnected proteins, ERH, engages in multiple RNA processes, including via interactions with nuclear speckles and the mRNA export machinery. We also demonstrate that the spliceosomal protein SNRNP200 participates in distinct stress granule-associated RNPs and occupies different RNA target regions in the cytoplasm during stress. Our comprehensive RBP-focused PPI network is a novel resource for identifying multi-stage RBPs and exploring RBP complexes in RNA maturation.

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“…As such, RBPs are crucial for guiding gene expression in time, space and depending on the cellular context. Hundreds of RBPs have been discovered so far (Hentze et al, 2018) and numerous methods have been developed to understand which RNAs are bound by which RBPs and where (Garriga-Canut et al, 2019;König et al, 2010;Lambert et al, 2014;Van Nostrand et al, 2016;Ray et al, 2009). The locations where RBPs bind an RNA are guided by primary RNA sequences (Ray et al, 2013), tertiary structures (Stefl et al, 2010;Tan et al, 2013) and post translational modifications (Zaccara et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

rec-Y2H matrix screening reveals a vast potential for direct protein-protein interactions among RNA binding proteins

Lang

Yang

Garriga-Canut

et al. 2020

Preprint

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RNA-binding proteins (RBPs) are crucial factors of post-transcriptional gene regulation and their modes of action are intensely investigated. At the center of attention are RNA motifs that guide where RBPs bind. However, sequence motifs are often poor predictors of RBP-RNA interactions in vivo. It is hence believed that many RBPs recognize RNAs as complexes, to increase specificity and regulatory potential. To probe the potential for complex formation among RBPs, we assembled a library of 978 mammalian RBPs and used rec-Y2H screening to detect direct interactions between RBPs, sampling >1M possible interactions. We discovered 1994 new interactions and demonstrate that our screening detects interactions between RBPs that bind RNA in proximity. We further find that the mRNA binding region preferences of an RBP can deviate, depending on its adjacently binding interaction partner. Finally, we reveal novel RBP interaction networks among major RNA processing steps and show that RBP mutations observed in cancer rewire spliceosomal interaction networks.

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rec-Y3H screening allows the detection of simultaneous RNA-protein interface mutations

Cited by 5 publications

References 20 publications

Mammalian Neuronal mRNA Transport Complexes: The Few Knowns and the Many Unknowns

Mammalian Neuronal mRNA Transport Complexes: The Few Knowns and the Many Unknowns

Large-scale map of RNA binding protein interactomes across the mRNA life-cycle

rec-Y2H matrix screening reveals a vast potential for direct protein-protein interactions among RNA binding proteins

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