RESUMO
Y-box-binding protein 1 (YB-1) is a multifunctional RNA binding protein involved in virtually every step of RNA metabolism. However, the functions and mechanisms of YB-1 in one of the most aggressive cancers, glioblastoma, are not well understood. In this study, we found that YB-1 protein was markedly overexpressed in glioblastoma and acted as a critical activator of both mTORC1 and mTORC2 signaling. Mechanistically, YB-1 bound the 5'UTR of CCT4 mRNA to promote the translation of CCT4, a component of the CCT chaperone complex, that in turn activated the mTOR signaling pathway by promoting mLST8 folding. In addition, YB-1 autoregulated its own translation by binding to its 5'UTR, leading to sustained activation of mTOR signaling. In patients with glioblastoma, high protein expression of YB-1 correlated with increased expression of CCT4 and mLST8 and activated mTOR signaling. Importantly, the administration of RNA decoys specifically targeting YB-1 in a mouse xenograft model resulted in slower tumor growth and better survival. Taken together, these findings uncover a disrupted proteostasis pathway involving a YB-1/CCT4/mLST8/mTOR axis in promoting glioblastoma growth, suggesting that YB-1 is a potential therapeutic target for the treatment of glioblastoma.
Assuntos
Glioblastoma , Proteína 1 de Ligação a Y-Box , Regiões 5' não Traduzidas , Animais , Linhagem Celular Tumoral , Chaperonina com TCP-1 , Glioblastoma/genética , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/genética , Proteína 1 de Ligação a Y-Box/genética , Proteína 1 de Ligação a Y-Box/metabolismo , Homólogo LST8 da Proteína Associada a mTOR/genética , Homólogo LST8 da Proteína Associada a mTOR/metabolismoRESUMO
BACKGROUND: Most of the microRNAs (MiRs) involved in myogenesis are transcriptional regulated. The role of MiR biogenesis in myogenesis has not been characterized yet. RNA-binding protein Musashi 2 (Msi2) is considered to be one of the major drivers for oncogenesis and stem cell proliferation. The functions of Msi2 in myogenesis have not been explored yet. We sought to investigate Msi2-regulated biogenesis of MiRs in myogenesis and muscle stem cell (MuSC) ageing. METHODS: We detected the expression of Msi2 in MuSCs and differentiated myotubes by quantitative reverse transcription PCR (RT-qPCR) and western blot. Msi2-binding partner human antigen R (HuR) was identified by immunoprecipitation followed by mass spectrometry analysis. The cooperative binding of Msi2 and HuR on MiR7a-1 was analysed by RNA immunoprecipitation and electrophoresis mobility shift assays. The inhibition of the processing of pri-MiR7a-1 mediated by Msi2 and HuR was shown by Msi2 and HuR knockdown. Immunofluorescent staining, RT-qPCR and immunoblotting were used to characterize the function of MiR7a-1 in myogenesis. Msi2 and HuR up-regulate cryptochrome circadian regulator 2 (Cry2) via MiR7a-1 was confirmed by the luciferase assay and western blot. The post-transcriptional regulatory cascade was further confirmed by RNAi and overexpressing of Msi2 and HuR in MuSCs, and the in vivo function was characterized by histopathological and molecular biological methods in Msi2 knockout mice. RESULTS: We identified a post-transcription regulatory cascade governed by a pair of RNA-binding proteins Msi2 and HuR. Msi2 is enriched in differentiated muscle cells and promotes MuSC differentiation despite its pro-proliferation functions in other cell types. Msi2 works synergistically with another RNA-binding protein HuR to repress the biogenesis of MiR7a-1 in an Msi2 dose-dependent manner to regulate the translation of the key component of the circadian core oscillator complex Cry2. Down-regulation of Cry2 (0.6-fold, vs. control, P < 0.05) mediated by MiR7a-1 represses MuSC differentiation. The disruption of this cascade leads to differentiation defects of MuSCs. In aged muscles, Msi2 (0.3-fold, vs. control, P < 0.01) expression declined, and the Cry2 protein level also decreases (0.5-fold, vs. control, P < 0.05), suggesting that the disruption of the Msi2-mediated post-transcriptional regulatory cascade could attribute to the declined ability of muscle regeneration in aged skeletal muscle. CONCLUSIONS: Our findings have identified a new post-transcriptional cascade regulating myogenesis. The cascade is disrupted in skeletal muscle ageing, which leads to declined muscle regeneration ability.
Assuntos
MicroRNAs , Desenvolvimento Muscular , Proteínas de Ligação a RNA/metabolismo , Animais , Diferenciação Celular/genética , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismoRESUMO
The congenital intellectual disability (ID)-causing gene mutations remain largely unclear, although many genetic variations might relate to ID. We screened gene mutations in Chinese Han children suffering from severe ID and found a single-nucleotide polymorphism (SNP) in the 5'-untranslated region (5'-UTR) of fibroblast growth factor 13 (FGF13) mRNA (NM_001139500.1:c.-32c>G) shared by three male children. In both HEK293 cells and patient-derived induced pluripotent stem cells, this SNP reduced the translation of FGF13, which stabilizes microtubules in developing neurons. Mice carrying the homologous point mutation in 5'-UTR of Fgf13 showed delayed neuronal migration during cortical development, and weakened learning and memory. Furthermore, this SNP reduced the interaction between FGF13 5'-UTR and polypyrimidine-tract-binding protein 2 (PTBP2), which was required for FGF13 translation in cortical neurons. Thus, this 5'-UTR SNP of FGF13 interferes with the translational process of FGF13 and causes deficits in brain development and cognitive functions.
Assuntos
Regiões 5' não Traduzidas/genética , Fatores de Crescimento de Fibroblastos/genética , Deficiência Intelectual/genética , Mutação Puntual , Polimorfismo de Nucleotídeo Único , Adolescente , Animais , Criança , Pré-Escolar , Fatores de Crescimento de Fibroblastos/metabolismo , Células HEK293 , Humanos , Aprendizagem , Masculino , Memória , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Accumulating evidence indicates that the alternative splicing program undergoes extensive changes during cancer development and progression. The RNA-binding protein QKI-5 is frequently downregulated and exhibits anti-tumor activity in lung cancer. Howeve-r, little is known about the functional targets and regulatory mechanism of QKI-5. Here, we report that upregulation of exon 14 inclusion of cytoskeletal gene Adducin 3 (ADD3) significantly correlates with a poor prognosis in lung cancer. QKI-5 inhibits cell proliferation and migration in part through suppressing the splicing of ADD3 exon 14. Through genome-wide mapping of QKI-5 binding sites in vivo at nucleotide resolution by iCLIP-seq analysis, we found that QKI-5 regulates alternative splicing of its target mRNAs in a binding position-dependent manner. By binding to multiple sites in an upstream intron region, QKI-5 represses the splicing of ADD3 exon 14. We also identified several QKI mutations in tumors, which cause dysregulation of the splicing of QKI targets ADD3 and NUMB. Taken together, our results reveal that QKI-mediated alternative splicing of ADD3 is a key lung cancer-associated splicing event, which underlies in part the tumor suppressor function of QKI.
Assuntos
Processamento Alternativo/genética , Proteínas de Ligação a Calmodulina/genética , Citoesqueleto/genética , Neoplasias Pulmonares/genética , Proteínas de Ligação a RNA/genética , Células A549 , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação para Baixo/genética , Éxons/genética , Genes Supressores de Tumor/fisiologia , Células HEK293 , Humanos , Íntrons/genética , Neoplasias Pulmonares/patologia , RNA Mensageiro/genética , Regulação para Cima/genéticaRESUMO
The Y-box binding protein 1 (YB-1) is a member of the cold shock domain (CSD) protein family and is recognized as an oncogenic factor in several solid tumors. By binding to RNA, YB-1 participates in several steps of posttranscriptional regulation of gene expression, including mRNA splicing, stability, and translation; microRNA processing; and stress granule assembly. However, the mechanisms in YB-1-mediated regulation of RNAs are unclear. Previously, we used both systematic evolution of ligands by exponential enrichment (SELEX) and individual-nucleotide resolution UV cross-linking and immunoprecipitation coupled RNA-Seq (iCLIP-Seq) analyses, which defined the RNA-binding consensus sequence of YB-1 as CA(U/C)C. We also reported that through binding to its core motif CAUC in primary transcripts, YB-1 regulates the alternative splicing of a CD44 variable exon and the biogenesis of miR-29b-2 during both Drosha and Dicer steps. To elucidate the molecular basis of the YB-1-RNA interactions, we report high-resolution crystal structures of the YB-1 CSD in complex with different RNA oligos at 1.7 Å resolution. The structure revealed that CSD interacts with RNA mainly through π-π stacking interactions assembled by four highly conserved aromatic residues. Interestingly, YB-1 CSD forms a homodimer in solution, and we observed that two residues, Tyr-99 and Asp-105, at the dimer interface are important for YB-1 CSD dimerization. Substituting these two residues with Ala reduced CSD's RNA-binding activity and abrogated the splicing activation of YB-1 targets. The YB-1 CSD-RNA structures presented here at atomic resolution provide mechanistic insights into gene expression regulated by CSD-containing proteins.
Assuntos
Proteína 1 de Ligação a Y-Box/metabolismo , Proteína 1 de Ligação a Y-Box/ultraestrutura , Processamento Alternativo/fisiologia , Proteínas de Ligação a DNA/metabolismo , Éxons/genética , Éxons/fisiologia , Humanos , Ligação Proteica , RNA/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/ultraestrutura , Proteína 1 de Ligação a Y-Box/genéticaRESUMO
RNA-binding proteins (RBPs) are key regulators in post-transcriptional control of gene expression. Mutations that alter their activity or abundance have been implicated in numerous diseases such as neurodegenerative disorders and various types of cancer. This highlights the importance of RBP proteostasis and the necessity to tightly control the expression levels and activities of RBPs. In many cases, RBPs engage in an auto-regulatory feedback by directly binding to and influencing the fate of their own mRNAs, exerting control over their own expression. For this feedback control, RBPs employ a variety of mechanisms operating at all levels of post-transcriptional regulation of gene expression. Here we review RBP-mediated autogenous feedback regulation that either serves to maintain protein abundance within a physiological range (by negative feedback) or generates binary, genetic on/off switches important for e.g. cell fate decisions (by positive feedback).
Assuntos
Proteínas de Ligação a RNA/metabolismo , Regulação da Expressão Gênica , Humanos , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
The histone H3K36 methyltransferase SETD2 is frequently mutated or deleted in a variety of human tumors. Nevertheless, the role of SETD2 loss in oncogenesis remains largely undefined. Here, we found that SETD2 counteracts Wnt signaling and its inactivation promotes intestinal tumorigenesis in mouse models of colorectal cancer (CRC). SETD2 was not required for intestinal homeostasis under steady state; however, upon irradiation, genetic inactivation of Setd2 in mouse intestinal epithelium facilitated the self-renewal of intestinal stem/progenitor cells as well as tissue regeneration. Furthermore, depletion of SETD2 enhanced the susceptibility to tumorigenesis in the context of dysregulated Wnt signaling. Mechanistic characterizations indicated that SETD2 downregulation affects the alternative splicing of a subset of genes implicated in tumorigenesis. Importantly, we uncovered that SETD2 ablation reduces intron retention of dishevelled segment polarity protein 2 (DVL2) pre-mRNA, which would otherwise be degraded by nonsense-mediated decay, thereby augmenting Wnt signaling. The signaling cascades mediated by SETD2 were further substantiated by a CRC patient cohort analysis. Together, our studies highlight SETD2 as an integral regulator of Wnt signaling through epigenetic regulation of RNA processing during tissue regeneration and tumorigenesis.
Assuntos
Processamento Alternativo , Carcinogênese/genética , Neoplasias Colorretais/genética , Histona-Lisina N-Metiltransferase/genética , Animais , Diferenciação Celular , Transformação Celular Neoplásica , Modelos Animais de Doenças , Proteínas Desgrenhadas/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Humanos , Íntrons , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Regeneração , Transdução de Sinais , Células-Tronco/citologia , Proteínas Wnt/metabolismoRESUMO
MicroRNA (miRNA) biogenesis is finely controlled by complex layers of post-transcriptional regulators, including RNA-binding proteins (RBPs). Here, we show that an RBP, QKI5, activates the processing of primary miR-124-1 (pri-124-1) during erythropoiesis. QKI5 recognizes a distal QKI response element and recruits Microprocessor through interaction with DGCR8. Furthermore, the recruited Microprocessor is brought to pri-124-1 stem loops by a spatial RNA-RNA interaction between two complementary sequences. Thus, mutations disrupting their base-pairing affect the strength of QKI5 activation. When erythropoiesis proceeds, the concomitant decrease of QKI5 releases Microprocessor from pri-124-1 and reduces mature miR-124 levels to facilitate erythrocyte maturation. Mechanistically, miR-124 targets TAL1 and c-MYB, two transcription factors involved in normal erythropoiesis. Importantly, this QKI5-mediated regulation also gives rise to a unique miRNA signature, which is required for erythroid differentiation. Taken together, these results demonstrate the pivotal role of QKI5 in primary miRNA processing during erythropoiesis and provide new insights into how a distal element on primary transcripts affects miRNA biogenesis.
Assuntos
Eritropoese/genética , MicroRNAs/genética , Motivos de Nucleotídeos/genética , Processamento Pós-Transcricional do RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA/genética , Animais , Sequência de Bases , Diferenciação Celular/genética , Células Eritroides/citologia , Células Eritroides/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Xenoenxertos , Humanos , Células K562 , Camundongos , MicroRNAs/metabolismo , Ligação Proteica/genética , Proteínas Proto-Oncogênicas c-myb , RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína 1 de Leucemia Linfocítica Aguda de Células T/metabolismoRESUMO
Extracellular signals have been shown to impact on alternative pre-mRNA splicing; however, the molecular mechanisms and biological significance of signal-induced splicing regulation remain largely unknown. Here, we report that epidermal growth factor (EGF) induces splicing changes through ubiquitylation of a well-known splicing regulator, hnRNP A1. EGF signaling upregulates an E3 ubiquitin (Ub) ligase adaptor, SPRY domain-containing SOCS box protein 1 (SPSB1), which recruits Elongin B/C-Cullin complexes to conjugate lysine 29-linked polyUb chains onto hnRNP A1. Importantly, SPSB1 and ubiquitylation of hnRNP A1 have a critical role in EGF-driven cell migration. Mechanistically, EGF-induced ubiquitylation of hnRNP A1 together with the activation of SR protein kinases (SRPKs) results in the upregulation of a Rac1 splicing isoform, Rac1b, to promote cell motility. These findings unravel a novel crosstalk between protein ubiquitylation and alternative splicing in EGF/EGF receptor signaling, and identify a new EGF/SPSB1/hnRNP A1/Rac1 axis in modulating cell migration, which may have important implications for cancer treatment.
Assuntos
Processamento Alternativo/genética , Movimento Celular , Fator de Crescimento Epidérmico/farmacologia , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Transdução de Sinais , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Ubiquitinação , Movimento Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proteínas Culina/metabolismo , Elonguina/metabolismo , Receptores ErbB/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisina/metabolismo , Poliubiquitina/metabolismo , Ligação Proteica/efeitos dos fármacos , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Alternative pre-mRNA splicing is a key mechanism for increasing proteomic diversity and modulating gene expression. Emerging evidence indicated that the splicing program is frequently dysregulated during tumorigenesis. Cancer cells produce protein isoforms that can promote growth and survival. The RNA-binding protein QKI5 is a critical regulator of alternative splicing in expanding lists of primary human tumors and tumor cell lines. However, its biological role and regulatory mechanism are poorly defined in gastric cancer (GC) development and progression. In this study, we demonstrated that the downregulation of QKI5 was associated with pTNM stage and pM state of GC patients. Re-introduction of QKI5 could inhibit GC cell proliferation, migration, and invasion in vitro and in vivo, which might be due to the altered splicing pattern of macroH2A1 pre-mRNA, leading to the accumulation of macroH2A1.1 isoform. Furthermore, QKI5 could inhibit cyclin L1 expression via promoting macroH2A1.1 production. Thus, this study identified a novel regulatory axis involved in gastric tumorigenesis and provided a new strategy for GC therapy.
Assuntos
Processamento Alternativo , Histonas/metabolismo , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Neoplasias Gástricas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Ciclinas/genética , Ciclinas/metabolismo , Feminino , Xenoenxertos , Histonas/genética , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Estadiamento de Neoplasias , Isoformas de Proteínas , Precursores de RNA/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Fatores de Tempo , Transfecção , Carga Tumoral , Proteínas Supressoras de Tumor/genéticaRESUMO
Altered miRNA expression is believed to play a crucial role in a variety of human cancers; however, the mechanisms leading to the dysregulation of miRNA expression remain elusive. In this study, we report that the human Y box-binding protein (YB-1), a major mRNA packaging protein, is a novel modulator of miRNA processing in glioblastoma multiforme (GBM). Using individual nucleotide-resolution crosslinking immunoprecipitation coupled to deep sequencing (iCLIP-seq), we performed the first genome-wide analysis of the in vivo YB-1-RNA interactions and found that YB-1 preferentially recognizes a UYAUC consensus motif and binds to the majority of coding gene transcripts including pre-mRNAs and mature mRNAs. Remarkably, our data show that YB-1 also binds extensively to the terminal loop region of pri-/pre-miR-29b-2 and regulates the biogenesis of miR-29b-2 by blocking the recruitment of microprocessor and Dicer to its precursors. Furthermore, we show that down-regulation of miR-29b by YB-1, which is up-regulated in GBM, is important for cell proliferation. Together, our findings reveal a novel function of YB-1 in regulating non-coding RNA expression, which has important implications in tumorigenesis.
Assuntos
Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA , Proteína 1 de Ligação a Y-Box/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células , Genoma Humano , Genômica , Glioblastoma/enzimologia , Glioblastoma/metabolismo , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , MicroRNAs/química , Ligação Proteica , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/antagonistas & inibidores , Análise de Sequência de RNARESUMO
BS69 (also called ZMYND11) contains tandemly arranged PHD, BROMO, and PWWP domains, which are chromatin recognition modalities. Here, we show that BS69 selectively recognizes histone variant H3.3 lysine 36 trimethylation (H3.3K36me3) via its chromatin-binding domains. We further identify BS69 association with RNA splicing regulators, including the U5 snRNP components of the spliceosome, such as EFTUD2. Remarkably, RNA sequencing shows that BS69 mainly regulates intron retention (IR), which is the least understood RNA alternative splicing event in mammalian cells. Biochemical and genetic experiments demonstrate that BS69 promotes IR by antagonizing EFTUD2 through physical interactions. We further show that regulation of IR by BS69 also depends on its binding to H3K36me3-decorated chromatin. Taken together, our study identifies an H3.3K36me3-specific reader and a regulator of IR and reveals that BS69 connects histone H3.3K36me3 to regulated RNA splicing, providing significant, important insights into chromatin regulation of pre-mRNA processing.
Assuntos
Processamento Alternativo , Proteínas de Transporte/metabolismo , Cromatina/metabolismo , Histonas/metabolismo , Precursores de RNA/genética , RNA Mensageiro/genética , Sequência de Bases , Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Cromatina/genética , Proteínas Correpressoras , Metilação de DNA/genética , Proteínas de Ligação a DNA , Células HeLa , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Humanos , Íntrons/genética , Lisina/genética , Lisina/metabolismo , Fatores de Alongamento de Peptídeos/antagonistas & inibidores , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , Processamento Pós-Transcricional do RNA/genética , RNA Interferente Pequeno , Ribonucleoproteína Nuclear Pequena U5/antagonistas & inibidores , Ribonucleoproteína Nuclear Pequena U5/genética , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Análise de Sequência de RNA , Spliceossomos/genéticaRESUMO
Lung cancer is the leading cause of cancer-related death worldwide. Aberrant splicing has been implicated in lung tumorigenesis. However, the functional links between splicing regulation and lung cancer are not well understood. Here we identify the RNA-binding protein QKI as a key regulator of alternative splicing in lung cancer. We show that QKI is frequently down-regulated in lung cancer, and its down-regulation is significantly associated with a poorer prognosis. QKI-5 inhibits the proliferation and transformation of lung cancer cells both in vitro and in vivo. Our results demonstrate that QKI-5 regulates the alternative splicing of NUMB via binding to two RNA elements in its pre-mRNA, which in turn suppresses cell proliferation and prevents the activation of the Notch signaling pathway. We further show that QKI-5 inhibits splicing by selectively competing with a core splicing factor SF1 for binding to the branchpoint sequence. Taken together, our data reveal QKI as a critical regulator of splicing in lung cancer and suggest a novel tumor suppression mechanism involving QKI-mediated regulation of the Notch signaling pathway.
Assuntos
Processamento Alternativo/genética , Neoplasias Pulmonares/genética , Splicing de RNA/genética , Proteínas de Ligação a RNA/genética , Proteínas Supressoras de Tumor/genética , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Regulação para Baixo/genética , Genes Supressores de Tumor , Humanos , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , RNA/genética , Precursores de RNA/genética , RNA Mensageiro/genética , Receptores Notch/genética , Transdução de Sinais/genéticaRESUMO
The human Y box-binding protein-1 (YB-1) is a deoxyribonucleic acid (DNA)/ribonucleic acid (RNA)-binding protein with pleiotropic functions. Besides its roles in the regulation of transcription and translation, several recent studies indicate that YB-1 is a spliceosome-associated protein and is involved in alternative splicing, but the underlying mechanism has remained elusive. Here, we define both CAUC and CACC as high-affinity binding motifs for YB-1 by systematic evolution of ligands by exponential enrichment (SELEX) and demonstrate that these newly defined motifs function as splicing enhancers. Interestingly, on the endogenous CD44 gene, YB-1 appears to mediate a network interaction to activate exon v5 inclusion via multiple CAUC motifs in both the alternative exon and its upstream polypyrimidine tract. We provide evidence that YB-1 activates splicing by facilitating the recruitment of U2AF65 to weak polypyrimidine tracts through direct protein-protein interactions. Together, these findings suggest a vital role of YB-1 in activating a subset of weak 3' splice sites in mammalian cells.