Nicole M. Martinez

All Publications

• Nano-Mod-Amp reveals RNA sequence, structural and cell type specific features of pseudouridylation by PUS7. bioRxiv : the preprint server for biology Rodell, R., Jain, R., Shenasa, H., Montes, M., Robalin, N., Prodic, S., Eyras, E., Martinez, N. M. 2025

  Abstract

  Pseudouridines are abundant mRNA modifications that can impact splicing, translation, and stability to tune gene expression. PUS7 is one of the major mRNA pseudouridine synthase whose dysregulation leads to neurodevelopmental disorders and cancer, underscoring the critical function of PUS7-dependent pseudouridines. Beyond a short and degenerate consensus sequence, the molecular mechanisms underlying PUS7-mediated pseudouridylation remain unknown. A lack of targeted, high-throughput pseudouridine detection methods limits simultaneous interrogation of PUS7 regulatory features across many experimental conditions. We developed novel Nanopore sequencing tools, including Nano-Mod-Amp, to reveal pseudouridine stoichiometry, its RNA structural context, and dependence on PUS7 levels at specific sites across biological conditions. We identified a novel RNA structural signature that is associated with more efficient mRNA modification by PUS7. Pseudouridines are largely responsive to modulations in PUS7 protein levels, demonstrating the regulatory potential of varying PUS7 levels across cellular conditions. Conversely, PUS7 activity is also regulated in a cell-type specific manner, independent of PUS7 expression levels in a manner consistent with regulation by RNA structure and RNA binding proteins. Together, we developed Nanopore sequencing tools and uncovered new mechanisms of PUS7 regulation with a framework that can be applied to other RNA-modifying enzymes to query the regulation of the epitranscriptome.

  View details for DOI 10.1101/2025.10.30.685621

  View details for PubMedID 41279304

  View details for PubMedCentralID PMC12636312

• Charting a Future for Sequencing RNA and Its Modifications: A New Era for Biology and Medicine National Academies of Sciences, Engineering, and Medicine Rodell, R., Jain, R., Shenasa, H., Montes, M., Robalin, N., Prodic, S., Eyras, E., Martinez, N. M. 2024

  View details for DOI 10.17226/27165

• Why U matters: detection and functions of pseudouridine modifications in mRNAs. Trends in biochemical sciences Rodell, R., Robalin, N., Martinez, N. M. 2023

  Abstract

  The uridine modifications pseudouridine (Psi), dihydrouridine, and 5-methyluridine are present in eukaryotic mRNAs. Many uridine-modifying enzymes are associated with human disease, underscoring the importance of uncovering the functions of uridine modifications in mRNAs. These modified uridines have chemical properties distinct from those of canonical uridines, which impact RNA structure and RNA-protein interactions. Psi, the most abundant of these uridine modifications, is present across (pre-)mRNAs. Recent work has shown that many Psis are present at intermediate to high stoichiometries that are likely conducive to function and at locations that are poised to influence pre-/mRNA processing. Technological innovations and mechanistic investigations are unveiling the functions of uridine modifications in pre-mRNA splicing, translation, and mRNA stability, which are discussed in this review.

  View details for DOI 10.1016/j.tibs.2023.10.008

  View details for PubMedID 38097411

• Rewriting the message: Harnessing RNA pseudouridylation to tackle disease. Molecular cell Montes, M., Martinez, N. M. 2023; 83 (4): 503-506

  Abstract

  Adachi etal.1 and Song etal.2 demonstrate the feasibility of engineering pseudouridylation at specific sites and its utility to correct disease-causing premature termination codons (PTCs) in human cells.

  View details for DOI 10.1016/j.molcel.2023.02.001

  View details for PubMedID 36804913

• Pseudouridine synthases modify human pre-mRNA co-transcriptionally and affect pre-mRNA processing. Molecular cell Martinez, N. M., Su, A., Burns, M. C., Nussbacher, J. K., Schaening, C., Sathe, S., Yeo, G. W., Gilbert, W. V. 2022; 82 (3): 645-659.e9

  Abstract

  Pseudouridine is a modified nucleotide that is prevalent in human mRNAs and is dynamically regulated. Here, we investigate when in their life cycle mRNAs become pseudouridylated to illuminate the potential regulatory functions of endogenous mRNA pseudouridylation. Using single-nucleotide resolution pseudouridine profiling on chromatin-associated RNA from human cells, we identified pseudouridines in nascent pre-mRNA at locations associated with alternatively spliced regions, enriched near splice sites, and overlapping hundreds of binding sites for RNA-binding proteins. In vitro splicing assays establish a direct effect of individual endogenous pre-mRNA pseudouridines on splicing efficiency. We validate hundreds of pre-mRNA sites as direct targets of distinct pseudouridine synthases and show that PUS1, PUS7, and RPUSD4-three pre-mRNA-modifying pseudouridine synthases with tissue-specific expression-control widespread changes in alternative pre-mRNA splicing and 3' end processing. Our results establish a vast potential for cotranscriptional pre-mRNA pseudouridylation to regulate human gene expression via alternative pre-mRNA processing.

  View details for DOI 10.1016/j.molcel.2021.12.023

  View details for PubMedID 35051350

  View details for PubMedCentralID PMC8859966

• Pseudouridine site assignment by high-throughput in vitro RNA pseudouridylation and sequencing. Methods in enzymology Martinez, N. M., Schaening-Burgos, C., Gilbert, W. V. 2021; 658: 277-310

  Abstract

  Pseudouridine (Ψ) is one of the most abundant modifications in cellular RNAs. High-throughput pseudouridine profiling of eukaryotic mRNAs from cells has revealed novel sites of modification across the transcriptome. Pseudouridine affects RNA structure and RNA-protein interactions with the potential to influence many steps of mRNA metabolism and thereby affect gene expression. Identifying the mechanisms by which individual pseudouridines sites are modified by pseudouridine synthases (PUS) will facilitate studies on the molecular functions of Ψ. Multiple pseudouridine synthases are expressed in all organisms and might direct pseudouridylation of diverse cellular RNAs, but the RNA targets of many enzymes and their specificity determinants remain to be defined. We developed a high-throughput in vitro pseudouridylation assay followed by sequencing that allows validation of candidate sites identified in cells, assignment of sites as direct targets of PUS and interrogation of the RNA sequence and structural features that direct modification. We also implemented an analysis pipeline to assign Ψ sites from these data, including an updated approach to peak-calling that accounts for noisy signal from low-abundance transcripts.

  View details for DOI 10.1016/bs.mie.2021.06.026

  View details for PubMedID 34517951

  View details for PubMedCentralID PMC9258999

• Investigating Pseudouridylation Mechanisms by High-Throughput in Vitro RNA Pseudouridylation and Sequencing. Methods in molecular biology (Clifton, N.J.) Martinez, N. M., Gilbert, W. V. 2021; 2298: 379-397

  Abstract

  Pseudouridine profiling has revealed many previously unknown sites of the RNA modification pseudouridine (Ψ) in cellular RNAs. All organisms express multiple pseudouridine synthases (PUS) whose RNA targets and mechanisms of targeting remain to be elucidated. Here, we describe a high-throughput in vitro pseudouridylation assay to interrogate pseudouridine status upon incubation with recombinant pseudouridine synthases (PUS) at thousands of RNA sequences of interest in parallel. This approach allows validation of sites provisionally identified in cells, identification of the direct targets of individual PUS, and interrogation of the determinants of target recognition including primary sequence and RNA secondary structure.

  View details for DOI 10.1007/978-1-0716-1374-0_22

  View details for PubMedID 34085256

  View details for PubMedCentralID 4224642

• Regulation and Function of RNA Pseudouridylation in Human Cells. Annual review of genetics Borchardt, E. K., Martinez, N. M., Gilbert, W. V. 2020; 54: 309-336

  Abstract

  Recent advances in pseudouridine detection reveal a complex pseudouridine landscape that includes messenger RNA and diverse classes of noncoding RNA in human cells. The known molecular functions of pseudouridine, which include stabilizing RNA conformations and destabilizing interactions with varied RNA-binding proteins, suggest that RNA pseudouridylation could have widespread effects on RNA metabolism and gene expression. Here, we emphasize how much remains to be learned about the RNA targets of human pseudouridine synthases, their basis for recognizing distinct RNA sequences, and the mechanisms responsible for regulated RNA pseudouridylation. We also examine the roles of noncoding RNA pseudouridylation in splicing and translation and point out the potential effects of mRNA pseudouridylation on protein production, including in the context of therapeutic mRNAs.

  View details for DOI 10.1146/annurev-genet-112618-043830

  View details for PubMedID 32870730

  View details for PubMedCentralID PMC8007080

• mRNA structure determines modification by pseudouridine synthase 1. Nature chemical biology Carlile, T. M., Martinez, N. M., Schaening, C., Su, A., Bell, T. A., Zinshteyn, B., Gilbert, W. V. 2019; 15 (10): 966-974

  Abstract

  Pseudouridine (Ψ) is a post-transcriptional RNA modification that alters RNA-RNA and RNA-protein interactions that affect gene expression. Messenger RNA pseudouridylation was recently discovered as a widespread and conserved phenomenon, but the mechanisms responsible for selective, regulated pseudouridylation of specific sequences within mRNAs were unknown. Here, we have revealed mRNA targets for five pseudouridine synthases and probed the determinants of mRNA target recognition by the predominant mRNA pseudouridylating enzyme, Pus1, by developing high-throughput kinetic analysis of pseudouridylation in vitro. Combining computational prediction and rational mutational analysis revealed an RNA structural motif that is both necessary and sufficient for mRNA pseudouridylation. Applying this structural context information predicted hundreds of additional mRNA targets that were pseudouridylated in vivo. These results demonstrate a structure-dependent mode of mRNA target recognition by a conserved pseudouridine synthase and implicate modulation of RNA structure as the probable mechanism to regulate mRNA pseudouridylation.

  View details for DOI 10.1038/s41589-019-0353-z

  View details for PubMedID 31477916

  View details for PubMedCentralID PMC6764900

• Pre-mRNA modifications and their role in nuclear processing. Quantitative biology (Beijing, China) Martinez, N. M., Gilbert, W. V. 2018; 6 (3): 210-227

  Abstract

  Cellular non-coding RNAs are extensively modified post-transcriptionally, with more than 100 chemically distinct nucleotides identified to date. In the past five years, new sequencing based methods have revealed widespread decoration of eukaryotic messenger RNA with diverse RNA modifications whose functions in mRNA metabolism are only beginning to be known.Since most of the identified mRNA modifying enzymes are present in the nucleus, these modifications have the potential to function in nuclear pre-mRNA processing including alternative splicing. Here we review recent progress towards illuminating the role of pre-mRNA modifications in splicing and highlight key areas for future investigation in this rapidly growing field.Future studies to identify which modifications are added to nascent pre-mRNA and to interrogate the direct effects of individual modifications are likely to reveal new mechanisms by which nuclear pre-mRNA processing is regulated.

  View details for DOI 10.1007/s40484-018-0147-4

  View details for PubMedID 30533247

  View details for PubMedCentralID PMC6284822

• Inhibition of Zinc-Dependent Histone Deacetylases with a Chemically Triggered Electrophile. ACS chemical biology Boskovic, Z. V., Kemp, M. M., Freedy, A. M., Viswanathan, V. S., Pop, M. S., Fuller, J. H., Martinez, N. M., Figueroa Lazú, S. O., Hong, J. A., Lewis, T. A., Calarese, D., Love, J. D., Vetere, A., Almo, S. C., Schreiber, S. L., Koehler, A. N. 2016; 11 (7): 1844-51

  Abstract

  Unbiased binding assays involving small-molecule microarrays were used to identify compounds that display unique patterns of selectivity among members of the zinc-dependent histone deacetylase family of enzymes. A novel, hydroxyquinoline-containing compound, BRD4354, was shown to preferentially inhibit activity of HDAC5 and HDAC9 in vitro. Inhibition of deacetylase activity appears to be time-dependent and reversible. Mechanistic studies suggest that the compound undergoes zinc-catalyzed decomposition to an ortho-quinone methide, which covalently modifies nucleophilic cysteines within the proteins. The covalent nature of the compound-enzyme interaction has been demonstrated in experiments with biotinylated probe compound and with electrospray ionization-mass spectrometry.

  View details for DOI 10.1021/acschembio.6b00012

  View details for PubMedID 27064299

• Position-dependent activity of CELF2 in the regulation of splicing and implications for signal-responsive regulation in T cells. RNA biology Ajith, S., Gazzara, M. R., Cole, B. S., Shankarling, G., Martinez, N. M., Mallory, M. J., Lynch, K. W. 2016; 13 (6): 569-81

  Abstract

  CELF2 is an RNA binding protein that has been implicated in developmental and signal-dependent splicing in the heart, brain and T cells. In the heart, CELF2 expression decreases during development, while in T cells CELF2 expression increases both during development and in response to antigen-induced signaling events. Although hundreds of CELF2-responsive splicing events have been identified in both heart and T cells, the way in which CELF2 functions has not been broadly investigated. Here we use CLIP-Seq to identified physical targets of CELF2 in a cultured human T cell line. By comparing the results with known functional targets of CELF2 splicing regulation from the same cell line we demonstrate a generalizable position-dependence of CELF2 activity that is consistent with previous mechanistic studies of individual CELF2 target genes in heart and brain. Strikingly, this general position-dependence is sufficient to explain the bi-directional activity of CELF2 on 2 T cell targets recently reported. Therefore, we propose that the location of CELF2 binding around an exon is a primary predictor of CELF2 function in a broad range of cellular contexts.

  View details for DOI 10.1080/15476286.2016.1176663

  View details for PubMedID 27096301

  View details for PubMedCentralID PMC4962813

• Convergence of Acquired Mutations and Alternative Splicing of CD19 Enables Resistance to CART-19 Immunotherapy CANCER DISCOVERY Sotillo, E., Barrett, D. M., Black, K. L., Bagashev, A., Oldridge, D., Wu, G., Sussman, R., Lanauze, C., Ruella, M., Gazzara, M. R., Martinez, N. M., Harrington, C. T., Chung, E. Y., Perazzelli, J., Hofmann, T. J., Maude, S. L., Raman, P., Barrera, A., Gill, S., Lacey, S. F., Melenhorst, J. J., Allman, D., Jacoby, E., Fry, T., Mackall, C., Barash, Y., Lynch, K. W., Maris, J. M., Grupp, S. A., Thomas-Tikhonenko, A. 2015; 5 (12): 1282-1295

  Abstract

  The CD19 antigen, expressed on most B-cell acute lymphoblastic leukemias (B-ALL), can be targeted with chimeric antigen receptor-armed T cells (CART-19), but relapses with epitope loss occur in 10% to 20% of pediatric responders. We detected hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some relapse samples. However, we also discovered alternatively spliced CD19 mRNA species, including one lacking exon 2. Pull-down/siRNA experiments identified SRSF3 as a splicing factor involved in exon 2 retention, and its levels were lower in relapsed B-ALL. Using genome editing, we demonstrated that exon 2 skipping bypasses exon 2 mutations in B-ALL cells and allows expression of the N-terminally truncated CD19 variant, which fails to trigger killing by CART-19 but partly rescues defects associated with CD19 loss. Thus, this mechanism of resistance is based on a combination of deleterious mutations and ensuing selection for alternatively spliced RNA isoforms.CART-19 yield 70% response rates in patients with B-ALL, but also produce escape variants. We discovered that the underlying mechanism is the selection for preexisting alternatively spliced CD19 isoforms with the compromised CART-19 epitope. This mechanism suggests a possibility of targeting alternative CD19 ectodomains, which could improve survival of patients with B-cell neoplasms.

  View details for DOI 10.1158/2159-8290.CD-15-1020

  View details for Web of Science ID 000368577500024

  View details for PubMedID 26516065

  View details for PubMedCentralID PMC4670800

• Widespread JNK-dependent alternative splicing induces a positive feedback loop through CELF2-mediated regulation of MKK7 during T-cell activation. Genes & development Martinez, N. M., Agosto, L., Qiu, J., Mallory, M. J., Gazzara, M. R., Barash, Y., Fu, X. D., Lynch, K. W. 2015; 29 (19): 2054-66

  Abstract

  Alternative splicing is prevalent among genes encoding signaling molecules; however, the functional consequence of differential isoform expression remains largely unknown. Here we demonstrate that, in response to T-cell activation, the Jun kinase (JNK) kinase MAP kinase kinase 7 (MKK7) is alternatively spliced to favor an isoform that lacks exon 2. This isoform restores a JNK-docking site within MKK7 that is disrupted in the larger isoform. Consistently, we show that skipping of MKK7 exon 2 enhances JNK pathway activity, as indicated by c-Jun phosphorylation and up-regulation of TNF-α. Moreover, this splicing event is itself dependent on JNK signaling. Thus, MKK7 alternative splicing represents a positive feedback loop through which JNK promotes its own signaling. We further show that repression of MKK7 exon 2 is dependent on the presence of flanking sequences and the JNK-induced expression of the RNA-binding protein CELF2, which binds to these regulatory elements. Finally, we found that ∼25% of T-cell receptor-mediated alternative splicing events are dependent on JNK signaling. Strikingly, these JNK-dependent events are also significantly enriched for responsiveness to CELF2. Together, our data demonstrate a widespread role for the JNK-CELF2 axis in controlling splicing during T-cell activation, including a specific role in propagating JNK signaling.

  View details for DOI 10.1101/gad.267245.115

  View details for PubMedID 26443849

  View details for PubMedCentralID PMC4604346

• Induced transcription and stability of CELF2 mRNA drives widespread alternative splicing during T-cell signaling. Proceedings of the National Academy of Sciences of the United States of America Mallory, M. J., Allon, S. J., Qiu, J., Gazzara, M. R., Tapescu, I., Martinez, N. M., Fu, X. D., Lynch, K. W. 2015; 112 (17): E2139-48

  Abstract

  Studies in several cell types have highlighted dramatic and diverse changes in mRNA processing that occur upon cellular stimulation. However, the mechanisms and pathways that lead to regulated changes in mRNA processing remain poorly understood. Here we demonstrate that expression of the splicing factor CELF2 (CUGBP, Elav-like family member 2) is regulated in response to T-cell signaling through combined increases in transcription and mRNA stability. Transcriptional induction occurs within 6 h of stimulation and is dependent on activation of NF-κB. Subsequently, there is an increase in the stability of the CELF2 mRNA that correlates with a change in CELF2 3'UTR length and contributes to the total signal-induced enhancement of CELF2 expression. Importantly, we uncover dozens of splicing events in cultured T cells whose changes upon stimulation are dependent on CELF2 expression, and provide evidence that CELF2 controls a similar proportion of splicing events during human thymic T-cell development. Taken together, these findings expand the physiologic impact of CELF2 beyond that previously documented in developing neuronal and muscle cells to T-cell development and function, identify unappreciated instances of alternative splicing in the human thymus, and uncover novel mechanisms for CELF2 regulation that may broadly impact CELF2 expression across diverse cell types.

  View details for DOI 10.1073/pnas.1423695112

  View details for PubMedID 25870297

  View details for PubMedCentralID PMC4418860

• Control of alternative splicing in immune responses: many regulators, many predictions, much still to learn. Immunological reviews Martinez, N. M., Lynch, K. W. 2013; 253 (1): 216-36

  Abstract

  Most mammalian pre-mRNAs are alternatively spliced in a manner that alters the resulting open reading frame. Consequently, alternative pre-mRNA splicing provides an important RNA-based layer of protein regulation and cellular function. The ubiquitous nature of alternative splicing coupled with the advent of technologies that allow global interrogation of the transcriptome have led to an increasing awareness of the possibility that widespread changes in splicing patterns contribute to lymphocyte function during an immune response. Indeed, a few notable examples of alternative splicing have clearly been demonstrated to regulate T-cell responses to antigen. Moreover, several proteins key to the regulation of splicing in T cells have recently been identified. However, much remains to be done to truly identify the spectrum of genes that are regulated at the level of splicing in immune cells and to determine how many of these are controlled by currently known factors and pathways versus unknown mechanisms. Here, we describe the proteins, pathways, and mechanisms that have been shown to regulate alternative splicing in human T cells and discuss what is and is not known about the genes regulated by such factors. Finally, we highlight unifying themes with regards to the mechanisms and consequences of alternative splicing in the adaptive immune system and give our view of important directions for future studies.

  View details for DOI 10.1111/imr.12047

  View details for PubMedID 23550649

  View details for PubMedCentralID PMC3621013

• Alternative splicing networks regulated by signaling in human T cells. RNA (New York, N.Y.) Martinez, N. M., Pan, Q., Cole, B. S., Yarosh, C. A., Babcock, G. A., Heyd, F., Zhu, W., Ajith, S., Blencowe, B. J., Lynch, K. W. 2012; 18 (5): 1029-40

  Abstract

  The formation and execution of a productive immune response requires the maturation of competent T cells and a robust change in cellular activity upon antigen challenge. Such changes in cellular function depend on regulated alterations to protein expression. Previous research has focused on defining transcriptional changes that regulate protein expression during T-cell maturation and antigen stimulation. Here, we globally analyze another critical process in gene regulation during T-cell stimulation, alternative splicing. Specifically, we use RNA-seq profiling to identify 178 exons in 168 genes that exhibit robust changes in inclusion in response to stimulation of a human T-cell line. Supporting an important role for the global coordination of alternative splicing following T-cell stimulation, these signal-responsive exons are significantly enriched in genes with functional annotations specifically related to immune response. The vast majority of these genes also exhibit differential alternative splicing between naive and activated primary T cells. Comparison of the responsiveness of splicing to various stimuli in the cultured and primary T cells further reveals at least three distinct networks of signal-induced alternative splicing events. Importantly, we find that each regulatory network is specifically associated with distinct sequence features, suggesting that they are controlled by independent regulatory mechanisms. These results thus provide a basis for elucidating mechanisms of signal pathway-specific regulation of alternative splicing during T-cell stimulation.

  View details for DOI 10.1261/rna.032243.112

  View details for PubMedID 22454538

  View details for PubMedCentralID PMC3334690

• A novel HDAC inhibitor with a hydroxy-pyrimidine scaffold. Bioorganic & medicinal chemistry letters Kemp, M. M., Wang, Q., Fuller, J. H., West, N., Martinez, N. M., Morse, E. M., Weïwer, M., Schreiber, S. L., Bradner, J. E., Koehler, A. N. 2011; 21 (14): 4164-9

  Abstract

  Histone deacetylases (HDACs) are enzymes involved in many important biological functions. They have been linked to a variety of cancers, psychiatric disorders, and other diseases. Since small molecules can serve as probes to study the relevant biological roles of HDACs, novel scaffolds are necessary to develop more efficient, selective drug candidates. Screening libraries of molecules may yield structurally diverse probes that bind these enzymes and modulate their functions in cells. Here we report a small molecule with a novel hydroxy-pyrimidine scaffold that inhibits multiple HDAC enzymes and modulates acetylation levels in cells. Analogs were synthesized in an effort to evaluate structure-activity relationships.

  View details for DOI 10.1016/j.bmcl.2011.05.098

  View details for PubMedID 21696956

  View details for PubMedCentralID PMC3248787