Douglas Porter

All Publications

• Glucose binds and activates NSUN2 to promote translation and epidermal differentiation. Nucleic acids research Miao, W., Porter, D. F., Li, Y., Meservey, L. M., Yang, Y. Y., Ma, C., Ferguson, I. D., Tien, V. B., Jack, T. M., Ducoli, L., Lopez-Pajares, V., Tao, S., Savage, P. B., Wang, Y., Khavari, P. A. 2024

  Abstract

  Elevations in intracellular glucose concentrations are essential for epithelial cell differentiation by mechanisms that are not fully understood. Glucose has recently been found to directly bind several proteins to alter their functions to enhance differentiation. Among the newly identified glucose-binding proteins is NSUN2, an RNA-binding protein that we identified as indispensable for epidermal differentiation. Glucose was found to bind conserved sequences within NSUN2, enhancing its binding to S-adenosyl-L-methionine and boosting its enzymatic activity. Additionally, glucose enhanced NSUN2's proximity to proteins involved in mRNA translation, with NSUN2 modulating global messenger RNA (mRNA) translation, particularly that of key pro-differentiation mRNAs containing m5C modifications, such as GRHL3. Glucose thus engages diverse molecular mechanisms beyond its energetic roles to facilitate cellular differentiation processes.

  View details for DOI 10.1093/nar/gkae1097

  View details for PubMedID 39565212

• Cell-type-specific interacting proteins collaborate to regulate the timing of Cyclin B protein expression in male meiotic prophase. Development (Cambridge, England) Baker, C. C., Gallicchio, L., Matias, N. R., Porter, D. F., Parsanian, L., Taing, E., Tam, C., Fuller, M. T. 2023

  Abstract

  During meiosis, germ cell and stage-specific components impose additional layers of regulation on the core cell cycle machinery to set up an extended G2 period termed meiotic prophase. In Drosophila males, meiotic prophase lasts 3.5 days, during which spermatocytes upregulate of over 1800 genes and grow 25-fold. Previous work showed that the cell cycle regulator Cyclin B (CycB) is subject to translational repression in immature spermatocytes, mediated by the RNA-binding protein Rbp4 and its partner Fest. Here we show that the spermatocyte-specific protein Lut is required for translational repression of cycB in an 8-hour window just before spermatocytes are fully mature. In males mutant for rbp4 or lut, spermatocytes enter and exit meiotic division 6-8 hours earlier than in wild-type. In addition, spermatocyte-specific isoforms of Syncrip (Syp) are required for expression of CycB protein in mature spermatocytes and normal entry into the meiotic divisions. Lut and Syp interact with Fest independent of RNA. Thus a set of spermatocyte-specific regulators choreograph the timing of expression of CycB protein during male meiotic prophase.

  View details for DOI 10.1242/dev.201709

  View details for PubMedID 37882771

• Glucose dissociates DDX21 dimers to regulate mRNA splicing and tissue differentiation. Cell Miao, W., Porter, D. F., Lopez-Pajares, V., Siprashvili, Z., Meyers, R. M., Bai, Y., Nguyen, D. T., Ko, L. A., Zarnegar, B. J., Ferguson, I. D., Mills, M. M., Jilly-Rehak, C. E., Wu, C., Yang, Y., Meyers, J. M., Hong, A. W., Reynolds, D. L., Ramanathan, M., Tao, S., Jiang, S., Flynn, R. A., Wang, Y., Nolan, G. P., Khavari, P. A. 2023; 186 (1): 80

  Abstract

  Glucose is a universal bioenergy source; however, its role in controlling protein interactions is unappreciated, as are its actions during differentiation-associated intracellular glucose elevation. Azido-glucose click chemistry identified glucose binding to a variety of RNA binding proteins (RBPs), including the DDX21 RNA helicase, which was found to be essential for epidermal differentiation. Glucose bound the ATP-binding domain of DDX21, altering protein conformation, inhibiting helicase activity, and dissociating DDX21 dimers. Glucose elevation during differentiation was associated with DDX21 re-localization from the nucleolus to the nucleoplasm where DDX21 assembled into larger protein complexes containing RNA splicing factors. DDX21 localized to specific SCUGSDGC motif in mRNA introns in a glucose-dependent manner and promoted the splicing of key pro-differentiation genes, including GRHL3, KLF4, OVOL1, and RBPJ. These findings uncover a biochemical mechanism of action for glucose in modulating the dimerization and function of an RNA helicase essential for tissue differentiation.

  View details for DOI 10.1016/j.cell.2022.12.004

  View details for PubMedID 36608661

• Analyzing RNA-Protein Interactions by Cross-Link Rates and CLIP-seq Libraries. Current protocols Porter, D. F., Garg, R. M., Meyers, R. M., Miao, W., Ducoli, L., Zarnegar, B. J., Khavari, P. A. 2023; 3 (1): e659

  Abstract

  UV cross-linking-based methods are the most common tool to explore in vivo RNA-protein interactions. UV cross-linking enables the freezing of direct interactions in the cell, which can then be mapped by high-throughput sequencing through a family of methods termed CLIP-seq. CLIP-seq measures the distribution of cross-link events by purifying a protein of interest and sequencing the covalently bound RNA fragments. However, there are disagreements and ambiguities as to which proteins are RNA-binding proteins and what interactions are significant as all proteins contact all RNAs at some frequency. Here we describe a protocol for both determining RNA-protein interactions through a combination of RNA library preparation and the measurement of absolute cross-link rates, which helps determine what proteins are RNA-binding proteins and what interactions are significant. This protocol, comprising an updated form of the easyCLIP protocol, describes guidelines for RNA library preparation, oligo and protein standard construction, and the measurement of cross-link rates. These methods are easily visualizable through their fluorescent labels and can be adapted to study RNA-binding properties of both functional, high affinity RNA-binding proteins, and the accidental RNA interactions of non-RNA-binding proteins. © 2023 Wiley Periodicals LLC. Basic Protocol 1: RNA library construction Basic Protocol 2: Determining UV cross-link rates Support Protocol 1: Cross-linking and lysing cells Support Protocol 2: Adapter preparation Support Protocol 3: Preparation of cross-linked RBP standard.

  View details for DOI 10.1002/cpz1.659

  View details for PubMedID 36705610

• PROBER identifies proteins associated with programmable sequence-specific DNA in living cells. Nature methods Mondal, S., Ramanathan, M., Miao, W., Meyers, R. M., Rao, D., Lopez-Pajares, V., Siprashvili, Z., Reynolds, D. L., Porter, D. F., Ferguson, I., Neela, P., Zhao, Y., Meservey, L. M., Guo, M., Yang, Y., Li, L., Wang, Y., Khavari, P. A. 2022; 19 (8): 959-968

  Abstract

  DNA-protein interactions mediate physiologic gene regulation and may be altered by DNA variants linked to polygenic disease. To enhance the speed and signal-to-noise ratio (SNR) in the identification and quantification of proteins associated with specific DNA sequences in living cells, we developed proximal biotinylation by episomal recruitment (PROBER). PROBER uses high-copy episomes to amplify SNR, and proximity proteomics (BioID) to identify the transcription factors and additional gene regulators associated with short DNA sequences of interest. PROBER quantified both constitutive and inducible association of transcription factors and corresponding chromatin regulators to target DNA sequences and binding quantitative trait loci due to single-nucleotide variants. PROBER identified alterations in regulator associations due to cancer hotspot mutations in the hTERT promoter, indicating that these mutations increase promoter association with specific gene activators. PROBER provides an approach to rapidly identify proteins associated with specific DNA sequences and their variants in living cells.

  View details for DOI 10.1038/s41592-022-01552-w

  View details for PubMedID 35927480

• Targeted Proteomic Approaches for Proteome-Wide Characterizations of the AMP-Binding Capacities of Kinases. Journal of proteome research Miao, W., Yin, J., Porter, D. F., Jiang, X., Khavari, P. A., Wang, Y. 2022

  Abstract

  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. Nature communications Porter, D. F., Miao, W., Yang, X., Goda, G. A., Ji, A. L., Donohue, L. K., Aleman, M. M., Dominguez, D., Khavari, P. A. 2021; 12 (1): 1569

  Abstract

  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 Medina-Munoz, H. C., Lapointe, C. P., Porter, D. F., Wickens, M. 2020; 117 (38): 23539–47

  Abstract

  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 Qiu, C., Dutcher, R. C., Porter, D. F., Arava, Y., Wickens, M., Hall, T. M. 2019

  Abstract

  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 Preston, M. A., Porter, D. F., Chen, F., Buter, N., Lapointe, C. P., Keles, S., Kimble, J., Wickens, M. 2019; 16 (5): 437-+

  View details for DOI 10.1038/s41592-019-0370-6

  View details for Web of Science ID 000466430000028

• Methods to study RNA-protein interactions (vol 16, pg 225, 2019) NATURE METHODS Ramanathan, M., Porter, D. F., Khavari, P. A. 2019; 16 (4): 351

  View details for DOI 10.1038/s41592-019-0366-2

  View details for Web of Science ID 000462620400034

• Methods to study RNA-protein interactions. Nature methods Ramanathan, M., Porter, D. F., Khavari, P. A. 2019; 16 (3): 225–34

  Abstract

  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 Porter, D. F., Prasad, A., Carrick, B. H., Kroll-Connor, P., Wickens, M., Kimble, J. 2019; 9 (1): 153–65

  Abstract

  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 CELL REPORTS Aoki, S. T., Porter, D. F., Prasad, A., Wickens, M., Bingman, C. A., Kimble, J. 2018; 23 (13): 3769–75

  Abstract

  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