RESUMEN
The production of a mature mRNA requires coordination of multiple processing steps, which ultimately control its content, localization, and stability. These steps include some of the largest macromolecular machines in the cell, which were, until recently, considered undruggable due to their biological complexity. Building from an expanded understanding of the underlying mechanisms that drive these processes, a new wave of therapeutics is seeking to target RNA processing. With a focus on impacting gene regulation at the RNA level, such modalities offer potential for sequence-specific resolution in drug design. Here, we review our current understanding of RNA-processing events and their role in gene regulation, with a focus on the therapeutic opportunities that have emerged within this landscape.
Asunto(s)
Oligonucleótidos Antisentido , Procesamiento Postranscripcional del ARN , Regulación de la Expresión Génica , Humanos , Oligonucleótidos Antisentido/uso terapéutico , ARN/genética , ARN MensajeroRESUMEN
Carbamoyl phosphate synthetase 1 (CPS1) catalyzes the first step in the ammonia-detoxifying urea cycle, converting ammonia to carbamoyl phosphate under physiologic conditions. In cancer, CPS1 overexpression supports pyrimidine synthesis to promote tumor growth in some cancer types, while in others CPS1 activity prevents the buildup of toxic levels of intratumoral ammonia to allow for sustained tumor growth. Targeted CPS1 inhibitors may, therefore, provide a therapeutic benefit for cancer patients with tumors overexpressing CPS1. Herein, we describe the discovery of small-molecule CPS1 inhibitors that bind to a previously unknown allosteric pocket to block ATP hydrolysis in the first step of carbamoyl phosphate synthesis. CPS1 inhibitors are active in cellular assays, blocking both urea synthesis and CPS1 support of the pyrimidine biosynthetic pathway, while having no activity against CPS2. These newly discovered CPS1 inhibitors are a first step toward providing researchers with valuable tools for probing CPS1 cancer biology.
Asunto(s)
Carbamoil-Fosfato Sintasa (Amoniaco)/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Piperidinas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Tiazoles/farmacología , Adenosina Trifosfato/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Regulación Alostérica/efectos de los fármacos , Carbamoil-Fosfato Sintasa (Amoniaco)/genética , Carbamoil-Fosfato Sintasa (Amoniaco)/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Humanos , Hidrólisis/efectos de los fármacos , Modelos Moleculares , Estructura Molecular , Piperidinas/química , Bibliotecas de Moléculas Pequeñas/química , Tiazoles/químicaRESUMEN
Hotspot mutations in splicing factor genes have been recently reported at high frequency in hematological malignancies, suggesting the importance of RNA splicing in cancer. We analyzed whole-exome sequencing data across 33 tumor types in The Cancer Genome Atlas (TCGA), and we identified 119 splicing factor genes with significant non-silent mutation patterns, including mutation over-representation, recurrent loss of function (tumor suppressor-like), or hotspot mutation profile (oncogene-like). Furthermore, RNA sequencing analysis revealed altered splicing events associated with selected splicing factor mutations. In addition, we were able to identify common gene pathway profiles associated with the presence of these mutations. Our analysis suggests that somatic alteration of genes involved in the RNA-splicing process is common in cancer and may represent an underappreciated hallmark of tumorigenesis.
Asunto(s)
Tasa de Mutación , Neoplasias/genética , Factores de Empalme de ARN/genética , Línea Celular Tumoral , Genes Supresores de Tumor , Humanos , Mutación con Pérdida de Función , Neoplasias/clasificación , Oncogenes , Empalme del ARN/genéticaRESUMEN
SF3B is a multi-protein complex essential for branch site (BS) recognition and selection during pre-mRNA splicing. Several splicing modulators with antitumor activity bind SF3B and thereby modulate splicing. Here we report the crystal structure of a human SF3B core in complex with pladienolide B (PB), a macrocyclic splicing modulator and potent inhibitor of tumor cell proliferation. PB stalls SF3B in an open conformation by acting like a wedge within a hinge, modulating SF3B's transition to the closed conformation needed to form the BS adenosine-binding pocket and stably accommodate the BS/U2 duplex. This work explains the structural basis for the splicing modulation activity of PB and related compounds, and reveals key interactions between SF3B and a common pharmacophore, providing a framework for future structure-based drug design.
Asunto(s)
Antineoplásicos/farmacología , Compuestos Epoxi/farmacología , Macrólidos/farmacología , Fosfoproteínas/metabolismo , Factores de Empalme de ARN/metabolismo , Empalme del ARN/efectos de los fármacos , Adenosina/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/metabolismo , Sitios de Unión , Proteínas Portadoras/metabolismo , Proliferación Celular/efectos de los fármacos , Diseño de Fármacos , Compuestos Epoxi/química , Compuestos Epoxi/metabolismo , Células HCT116 , Células HeLa , Humanos , Macrólidos/química , Macrólidos/metabolismo , Modelos Moleculares , Complejos Multiproteicos , Fosfoproteínas/química , Fosfoproteínas/genética , Unión Proteica , Conformación Proteica , Precursores del ARN/genética , Precursores del ARN/metabolismo , Factores de Empalme de ARN/química , Factores de Empalme de ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN , Células Sf9 , Relación Estructura-Actividad , TransactivadoresRESUMEN
Genomic analyses of cancer have identified recurrent point mutations in the RNA splicing factor-encoding genes SF3B1, U2AF1, and SRSF2 that confer an alteration of function. Cancer cells bearing these mutations are preferentially dependent on wild-type (WT) spliceosome function, but clinically relevant means to therapeutically target the spliceosome do not currently exist. Here we describe an orally available modulator of the SF3b complex, H3B-8800, which potently and preferentially kills spliceosome-mutant epithelial and hematologic tumor cells. These killing effects of H3B-8800 are due to its direct interaction with the SF3b complex, as evidenced by loss of H3B-8800 activity in drug-resistant cells bearing mutations in genes encoding SF3b components. Although H3B-8800 modulates WT and mutant spliceosome activity, the preferential killing of spliceosome-mutant cells is due to retention of short, GC-rich introns, which are enriched for genes encoding spliceosome components. These data demonstrate the therapeutic potential of splicing modulation in spliceosome-mutant cancers.
Asunto(s)
Neoplasias/tratamiento farmacológico , Neoplasias/genética , Piperazinas/farmacología , Piridinas/farmacología , Empalme del ARN/genética , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Empalmosomas/genética , Administración Oral , Animales , Secuencia de Bases , Humanos , Intrones/genética , Células K562 , Leucemia/genética , Leucemia/patología , Ratones , Mutación , Neoplasias/patología , Piperazinas/administración & dosificación , Piridinas/administración & dosificación , Empalme del ARN/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Recently splicing has been recognized as a key pathway in cancer. Although aberrant splicing has been shown to be a consequence of mutations or the abnormal expression of splicing factors (trans-effect changes) or mutations in the splicing sequences (cis-effect mutations), the connections between aberrant splicing and cancer initiation or progression are still not well understood. Here we review the mutational landscape of splicing factors in cancer and associated splicing consequences, along with the most important examples of the therapeutic approaches targeting the spliceosome currently being investigated in oncology.
Asunto(s)
Neoplasias/tratamiento farmacológico , Neoplasias/genética , Oligonucleótidos/uso terapéutico , Empalme del ARN/efectos de los fármacos , Animales , Humanos , Mutación , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , Empalmosomas/efectos de los fármacos , Empalmosomas/metabolismoRESUMEN
Acquired point mutations of pre-mRNA splicing factors recur among cancers, leukemias, and related neoplasms. Several studies have established that somatic mutations of a U2AF1 subunit, which normally recognizes 3' splice site junctions, recur among myelodysplastic syndromes. The U2AF2 splicing factor recognizes polypyrimidine signals that precede most 3' splice sites as a heterodimer with U2AF1. In contrast with those of the well-studied U2AF1 subunit, descriptions of cancer-relevant U2AF2 mutations and their structural relationships are lacking. Here, we survey databases of cancer-associated mutations and identify recurring missense mutations in the U2AF2 gene. We determine ultra-high-resolution structures of the U2AF2 RNA recognition motifs (RRM1 and RRM2) at 1.1 Å resolution and map the structural locations of the mutated U2AF2 residues. Comparison with prior, lower-resolution structures of the tandem U2AF2 RRMs in the RNA-bound and apo states reveals clusters of cancer-associated mutations at the U2AF2 RRM-RNA or apo-RRM1-RRM2 interfaces. Although the role of U2AF2 mutations in malignant transformation remains uncertain, our results show that cancer-associated mutations correlate with functionally important surfaces of the U2AF2 splicing factor.
Asunto(s)
Neoplasias/metabolismo , ARN/metabolismo , Factor de Empalme U2AF/química , Factor de Empalme U2AF/metabolismo , Secuencias de Aminoácidos , Sitios de Unión , Cristalización , Humanos , Modelos Moleculares , Mutación , Conformación Proteica , Subunidades de ProteínaRESUMEN
We identify and characterize novel SF3B1 in-frame deletions in chronic lymphocytic leukemia.These deletions are functionally similar to well-known SF3B1 hotspot mutations and are sensitive to splicing modulation.
RESUMEN
How the essential pre-mRNA splicing factor U2AF(65) recognizes the polypyrimidine (Py) signals of the major class of 3' splice sites in human gene transcripts remains incompletely understood. We determined four structures of an extended U2AF(65)-RNA-binding domain bound to Py-tract oligonucleotides at resolutions between 2.0 and 1.5 Å. These structures together with RNA binding and splicing assays reveal unforeseen roles for U2AF(65) inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract. The U2AF(65) linker residues between the dual RNA recognition motifs (RRMs) recognize the central nucleotide, whereas the N- and C-terminal RRM extensions recognize the 3' terminus and third nucleotide. Single-molecule FRET experiments suggest that conformational selection and induced fit of the U2AF(65) RRMs are complementary mechanisms for Py-tract association. Altogether, these results advance the mechanistic understanding of molecular recognition for a major class of splice site signals.
Asunto(s)
Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Sitios de Empalme de ARN , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Cristalografía por Rayos X , Humanos , Proteínas Nucleares/genética , Estructura Terciaria de Proteína , Precursores del ARN/química , Precursores del ARN/genética , Precursores del ARN/metabolismo , Empalme del ARN , Ribonucleoproteínas/genética , Factor de Empalme U2AFRESUMEN
Recurrent mutations in the spliceosome are observed in several human cancers, but their functional and therapeutic significance remains elusive. SF3B1, the most frequently mutated component of the spliceosome in cancer, is involved in the recognition of the branch point sequence (BPS) during selection of the 3' splice site (ss) in RNA splicing. Here, we report that common and tumor-specific splicing aberrations are induced by SF3B1 mutations and establish aberrant 3' ss selection as the most frequent splicing defect. Strikingly, mutant SF3B1 utilizes a BPS that differs from that used by wild-type SF3B1 and requires the canonical 3' ss to enable aberrant splicing during the second step. Approximately 50% of the aberrantly spliced mRNAs are subjected to nonsense-mediated decay resulting in downregulation of gene and protein expression. These findings ascribe functional significance to the consequences of SF3B1 mutations in cancer.
Asunto(s)
Empalme Alternativo , Mutación , Neoplasias/genética , Fosfoproteínas/genética , Ribonucleoproteína Nuclear Pequeña U2/genética , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , Células HEK293 , Humanos , Datos de Secuencia Molecular , Tasa de Mutación , Degradación de ARNm Mediada por Codón sin Sentido , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Factores de Empalme de ARN , Ribonucleoproteína Nuclear Pequeña U2/química , Ribonucleoproteína Nuclear Pequeña U2/metabolismoRESUMEN
Purine interruptions of polypyrimidine (Py) tract splice site signals contribute to human genetic diseases. The essential splicing factor U2AF(65) normally recognizes a Py tract consensus sequence preceding the major class of 3' splice sites. We found that neurofibromatosis- or retinitis pigmentosa-causing mutations in the 5' regions of Py tracts severely reduce U2AF(65) affinity. Conversely, we identified a preferred binding site of U2AF(65) for purine substitutions in the 3' regions of Py tracts. Based on a comparison of new U2AF(65) structures bound to either A- or G-containing Py tracts with previously identified pyrimidine-containing structures, we expected to find that a D231V amino acid change in U2AF(65) would specify U over other nucleotides. We found that the crystal structure of the U2AF(65)-D231V variant confirms favorable packing between the engineered valine and a target uracil base. The D231V amino acid change restores U2AF(65) affinity for two mutated splice sites that cause human genetic diseases and successfully promotes splicing of a defective retinitis pigmentosa-causing transcript. We conclude that reduced U2AF(65) binding is a molecular consequence of disease-relevant mutations, and that a structure-guided U2AF(65) variant is capable of manipulating gene expression in eukaryotic cells.
Asunto(s)
Empalme Alternativo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Adenina/química , Sitios de Unión , Cristalografía por Rayos X , Escherichia coli/metabolismo , Variación Genética , Guanina/química , Humanos , Conformación Molecular , Mutación , Unión Proteica , Ingeniería de Proteínas , ARN/química , Factor de Empalme U2AF , Uracilo/químicaRESUMEN
Degenerate splice site sequences mark the intron boundaries of pre-mRNA transcripts in multicellular eukaryotes. The essential pre-mRNA splicing factor U2AF(65) is faced with the paradoxical tasks of accurately targeting polypyrimidine (Py) tracts preceding 3' splice sites while adapting to both cytidine and uridine nucleotides with nearly equivalent frequencies. To understand how U2AF(65) recognizes degenerate Py tracts, we determined six crystal structures of human U2AF(65) bound to cytidine-containing Py tracts. As deoxy-ribose backbones were required for co-crystallization with these Py tracts, we also determined two baseline structures of U2AF(65) bound to the deoxy-uridine counterparts and compared the original, RNA-bound structure. Local structural changes suggest that the N-terminal RNA recognition motif 1 (RRM1) is more promiscuous for cytosine-containing Py tracts than the C-terminal RRM2. These structural differences between the RRMs were reinforced by the specificities of wild-type and site-directed mutant U2AF(65) for region-dependent cytosine- and uracil-containing RNA sites. Small-angle X-ray scattering analyses further demonstrated that Py tract variations select distinct inter-RRM spacings from a pre-existing ensemble of U2AF(65) conformations. Our results highlight both local and global conformational selection as a means for universal 3' splice site recognition by U2AF(65).