Targeted Proteomic Approaches for Proteome-Wide Characterizations of the AMP-Binding Capacities of Kinases.
Journal of proteome research
Kinases play important roles in cell signaling, and adenosine monophosphate (AMP) is known to modulate cellular energy homeostasis through AMP-activated protein kinase (AMPK). Here, we explored novel AMP-binding kinases by employing a desthiobiotin-conjugated AMP acyl-phosphate probe to enrich efficiently AMP-binding proteins. Together with a parallel-reaction monitoring-based targeted proteomic approach, we uncovered 195 candidate AMP-binding kinases. We also enriched desthiobiotin-labeled peptides from adenine nucleotide-binding sites of kinases and analyzed them using LC-MS/MS in the multiple-reaction monitoring mode, which resulted in the identification of 44 peptides derived from 43 kinases displaying comparable or better binding affinities toward AMP relative to adenosine triphosphate (ATP). Moreover, our proteomic data revealed a potential involvement of AMP in the MAPK pathway through binding directly to the relevant kinases, especially MEK2 and MEK3. Together, we revealed the AMP-binding capacities of a large number of kinases, and our work built a strong foundation for understanding how AMP functions as a second messenger to modulate cell signaling.
View details for DOI 10.1021/acs.jproteome.2c00225
View details for PubMedID 35820187
easyCLIP analysis of RNA-protein interactions incorporating absolute quantification.
2021; 12 (1): 1569
Quantitative criteria to identify proteins as RNA-binding proteins (RBPs) are presently lacking, as are criteria to define RBP target RNAs. Here, we develop an ultraviolet (UV) cross-linking immunoprecipitation (CLIP)-sequencing method, easyCLIP. easyCLIP provides absolute cross-link rates, as well as increased simplicity, efficiency, and capacity to visualize RNA libraries during sequencing library preparation. Measurement of >200 independent cross-link experiments across >35 proteins identifies an RNA cross-link rate threshold that distinguishes RBPs from non-RBPs and defines target RNAs as those with a complex frequency unlikely for a random protein. We apply easyCLIP to the 33 most recurrent cancer mutations across 28 RBPs, finding increased RNA binding per RBP molecule for KHDRBS2 R168C, A1CF E34K and PCBP1 L100P/Q cancer mutations. Quantitating RBP-RNA interactions can thus nominate proteins as RBPs and define the impact of specific disease-associated RBP mutations on RNA association.
View details for DOI 10.1038/s41467-021-21623-4
View details for PubMedID 33692367
Records of RNA locations in living yeast revealed through covalent marks
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2020; 117 (38): 23539–47
RNA movements and localization pervade biology, from embryonic development to disease. To identify RNAs at specific locations, we developed a strategy in which a uridine-adding enzyme is anchored to subcellular sites, where it directly marks RNAs with 3' terminal uridines. This localized RNA recording approach yields a record of RNA locations, and is validated through identification of RNAs localized selectively to the endoplasmic reticulum (ER) or mitochondria. We identify a broad dual localization pattern conserved from yeast to human cells, in which the same battery of mRNAs encounter both ER and mitochondria in both species, and include an mRNA encoding a key stress sensor. Subunits of many multiprotein complexes localize to both the ER and mitochondria, suggesting coordinated assembly. Noncoding RNAs in the course of RNA surveillance and processing encounter both organelles. By providing a record of RNA locations over time, the approach complements those that capture snapshots of instantaneous positions.
View details for DOI 10.1073/pnas.1921408117
View details for Web of Science ID 000575888700022
View details for PubMedID 32907940
View details for PubMedCentralID PMC7519331
Distinct RNA-binding modules in a single PUF protein cooperate to determine RNA specificity.
Nucleic acids research
PUF proteins, named for Drosophila Pumilio (PUM) and Caenorhabditis elegans fem-3-binding factor (FBF), recognize specific sequences in the mRNAs they bind and control. RNA binding by classical PUF proteins is mediated by a characteristic PUM homology domain (PUM-HD). The Puf1 and Puf2 proteins possess a distinct architecture and comprise a highly conserved subfamily among fungal species. Puf1/Puf2 proteins contain two types of RNA-binding domain: a divergent PUM-HD and an RNA recognition motif (RRM). They recognize RNAs containing UAAU motifs, often in clusters. Here, we report a crystal structure of the PUM-HD of a fungal Puf1 in complex with a dual UAAU motif RNA. Each of the two UAAU tetranucleotides are bound by a Puf1 PUM-HD forming a 2:1 protein-to-RNA complex. We also determined crystal structures of the Puf1 RRM domain that identified a dimerization interface. The PUM-HD and RRM domains act in concert to determine RNA-binding specificity: the PUM-HD dictates binding to UAAU, and dimerization of the RRM domain favors binding to dual UAAU motifs rather than a single UAAU. Cooperative action of the RRM and PUM-HD identifies a new mechanism by which multiple RNA-binding modules in a single protein collaborate to create a unique RNA-binding specificity.
View details for DOI 10.1093/nar/gkz583
View details for PubMedID 31294800
- Unbiased screen of RNA tailing activities reveals a poly(UG) polymerase NATURE METHODS 2019; 16 (5): 437-+
- Methods to study RNA-protein interactions (vol 16, pg 225, 2019) NATURE METHODS 2019; 16 (4): 351
Methods to study RNA-protein interactions.
2019; 16 (3): 225–34
Noncoding RNA sequences, including long noncoding RNAs, small nucleolar RNAs, and untranslated mRNA regions, accomplish many of their diverse functions through direct interactions with RNA-binding proteins (RBPs). Recent efforts have identified hundreds of new RBPs that lack known RNA-binding domains, thus underscoring the complexity and diversity of RNA-protein complexes. Recent progress has expanded the number of methods for studying RNA-protein interactions in two general categories: approaches that characterize proteins bound to an RNA of interest (RNA-centric), and those that examine RNAs bound to a protein of interest (protein-centric). Each method has unique strengths and limitations, which makes it important to select optimal approaches for the biological question being addressed. Here we review methods for the study of RNA-protein interactions, with a focus on their suitability for specific applications.
View details for PubMedID 30804549
Toward Identifying Subnetworks from FBF Binding Landscapes in Caenorhabditis Spermatogenic or Oogenic Germlines
G3-GENES GENOMES GENETICS
2019; 9 (1): 153–65
Metazoan PUF (Pumilio and FBF) RNA-binding proteins regulate various biological processes, but a common theme across phylogeny is stem cell regulation. In Caenorhabditis elegans, FBF (fem-3 Binding Factor) maintains germline stem cells regardless of which gamete is made, but FBF also functions in the process of spermatogenesis. We have begun to "disentangle" these biological roles by asking which FBF targets are gamete-independent, as expected for stem cells, and which are gamete-specific. Specifically, we compared FBF iCLIP binding profiles in adults making sperm to those making oocytes. Normally, XX adults make oocytes. To generate XX adults making sperm, we used a fem-3(gf) mutant requiring growth at 25°; for comparison, wild-type oogenic hermaphrodites were also raised at 25°. Our FBF iCLIP data revealed FBF binding sites in 1522 RNAs from oogenic adults and 1704 RNAs from spermatogenic adults. More than half of these FBF targets were independent of germline gender. We next clustered RNAs by FBF-RNA complex frequencies and found four distinct blocks. Block I RNAs were enriched in spermatogenic germlines, and included validated target fog-3, while Block II and III RNAs were common to both genders, and Block IV RNAs were enriched in oogenic germlines. Block II (510 RNAs) included almost all validated FBF targets and was enriched for cell cycle regulators. Block III (21 RNAs) was enriched for RNA-binding proteins, including previously validated FBF targets gld-1 and htp-1 We suggest that Block I RNAs belong to the FBF network for spermatogenesis, and that Blocks II and III are associated with stem cell functions.
View details for DOI 10.1534/g3.118.200300
View details for Web of Science ID 000455206600015
View details for PubMedID 30459181
View details for PubMedCentralID PMC6325917
An RNA-Binding Multimer Specifies Nematode Sperm Fate
2018; 23 (13): 3769–75
FOG-3 is a master regulator of sperm fate in Caenorhabditis elegans and homologous to Tob/BTG proteins, which in mammals are monomeric adaptors that recruit enzymes to RNA binding proteins. Here, we determine the FOG-3 crystal structure and in vitro demonstrate that FOG-3 forms dimers that can multimerize. The FOG-3 multimeric structure has a basic surface potential, suggestive of binding nucleic acid. Consistent with that prediction, FOG-3 binds directly to nearly 1,000 RNAs in nematode spermatogenic germ cells. Most binding is to the 3' UTR, and most targets (94%) are oogenic mRNAs, even though assayed in spermatogenic cells. When tethered to a reporter mRNA, FOG-3 represses its expression. Together these findings elucidate the molecular mechanism of sperm fate specification and reveal the evolution of a protein from monomeric to multimeric form with acquisition of a distinct mode of mRNA repression.
View details for DOI 10.1016/j.celrep.2018.05.095
View details for Web of Science ID 000436517100009
View details for PubMedID 29949762