RESUMO
CDK11 is an emerging druggable target for cancer therapy due to its prevalent roles in phosphorylating critical transcription and splicing factors and in facilitating cell cycle progression in cancer cells. Like other cyclin-dependent kinases, CDK11 requires its cognate cyclin, cyclin L1 or cyclin L2, for activation. However, little is known about how CDK11 activities might be modulated by other regulators. In this study, we show that CDK11 forms a tight complex with cyclins L1/L2 and SAP30BP, the latter of which is a poorly characterized factor. Acute degradation of SAP30BP mirrors that of CDK11 in causing widespread and strong defects in pre-mRNA splicing. Furthermore, we demonstrate that SAP30BP facilitates CDK11 kinase activities in vitro and in vivo, through ensuring the stabilities and the assembly of cyclins L1/L2 with CDK11. Together, these findings uncover SAP30BP as a critical CDK11 activator that regulates global pre-mRNA splicing.
Assuntos
Precursores de RNA , Splicing de RNA , Precursores de RNA/genética , Precursores de RNA/metabolismo , Fosforilação , Divisão Celular , Ciclinas/genética , Ciclinas/metabolismoRESUMO
The RNA-binding protein ALYREF plays key roles in nuclear export and also 3'-end processing of polyadenylated mRNAs, but whether such regulation also extends to non-polyadenylated RNAs is unknown. Replication-dependent (RD)-histone mRNAs are not polyadenylated, but instead end in a stem-loop (SL) structure. Here, we demonstrate that ALYREF prevalently binds a region next to the SL on RD-histone mRNAs. SL-binding protein (SLBP) directly interacts with ALYREF and promotes its recruitment. ALYREF promotes histone pre-mRNA 3'-end processing by facilitating U7-snRNP recruitment through physical interaction with the U7-snRNP-specific component Lsm11. Furthermore, ALYREF, together with other components of the TREX complex, enhances histone mRNA export. Moreover, we show that 3'-end processing promotes ALYREF recruitment and histone mRNA export. Together, our results point to an important role of ALYREF in coordinating 3'-end processing and nuclear export of non-polyadenylated mRNAs.
Assuntos
Histonas/metabolismo , Proteínas Nucleares/metabolismo , Processamento Pós-Transcricional do RNA , Transporte de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U7/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Histonas/genética , Humanos , Proteínas Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Ribonucleoproteína Nuclear Pequena U7/genética , Fatores de Transcrição/genéticaRESUMO
To ensure efficient and accurate gene expression, pre-mRNA processing and mRNA export need to be balanced. However, how this balance is ensured remains largely unclear. Here, we found that SF3b, a component of U2 snRNP that participates in splicing and 3' processing of pre-mRNAs, interacts with the key mRNA export adaptor THO in vivo and in vitro. Depletion of SF3b reduces THO binding with the mRNA and causes nuclear mRNA retention. Consistently, introducing SF3b binding sites into the mRNA enhances THO recruitment and nuclear export in a dose-dependent manner. These data demonstrate a role of SF3b in promoting mRNA export. In support of this role, SF3b binds with mature mRNAs in the cells. Intriguingly, disruption of U2 snRNP by using a U2 antisense morpholino oligonucleotide does not inhibit, but promotes, the role of SF3b in mRNA export as a result of enhanced SF3b-THO interaction and THO recruitment to the mRNA. Together, our study uncovers a U2-snRNP-independent role of SF3b in mRNA export and suggests that SF3b contributes to balancing pre-mRNA processing and mRNA export.
Assuntos
Fosfoproteínas , Precursores de RNA , Fatores de Processamento de RNA , RNA Mensageiro , Ribonucleoproteína Nuclear Pequena U2 , Sítios de Ligação/genética , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismoRESUMO
A successful pregnancy depends on the blastocyst's implantation to the maternal endometrium; however, the initial interaction between blastocyst and uterine epithelium has not been well characterized. The objectives of this study were to determine if selectins and their ligands were expressed in the bovine conceptus and/or uterus during the periattachment period and to study whether selectins were associated with conceptus attachment to the uterine epithelium. Through the RNA-sequence analysis of bovine conceptuses on Days 17, 20, and 22 (Day 0 = day of estrus), only the SELL ligand, podocalyxin (PODXL), and P-selectin (SELP) ligand, SELPLG, were found. Quantitative PCR analysis confirmed the presence of PODXL and SELPLG in these conceptuses and revealed that SELL, mRNA and protein, detected in the uterine epithelium but not in conceptuses increased during the periattachment period. In the cultured endometrial epithelial cells (EECs), SELL transcript was up-regulated when uterine flushings from Day 20 pregnant animals were placed onto these cells. SELL was also up-regulated when cultured EECs were treated with progesterone, EGF, or bFGF, but not with IFNT. In the coculture system with EECs and bovine trophoblast CT-1 cells, SELL expression in EECs was effectively reduced by its small interfering RNA; however, IFNT, a marker for CT-1 cell attachment to EECs, was not reduced, nor was a transcription factor of IFNT, CDX2. These observations suggest that the conceptus could attach to the uterine epithelium through the use of endometrial SELL and embryonic selectin ligands, possibly initiating the conceptus attachment process in the bovine species.
Assuntos
Implantação do Embrião/fisiologia , Endométrio/fisiologia , Epitélio/fisiologia , Feto/fisiologia , Selectinas/fisiologia , Útero/fisiologia , Animais , Fator de Transcrição CDX2 , Bovinos , Células Cultivadas , Feminino , Proteínas de Homeodomínio/genética , Interferon Tipo I/genética , Ligantes , Glicoproteínas de Membrana/genética , Gravidez , Proteínas da Gravidez/genética , RNA/genética , RNA Interferente Pequeno/genética , Transativadores/genéticaRESUMO
In order to explore the practical application of ultrasonic imaging in the pregnancy stage of Mongolian sheep and the role of L-selectin in the embryo implantation process of Mongolian sheep, this paper systematically observed the early embryonic development by B-mode ultrasonic imaging wave diagnostic instrument with 5 MHz rectal probe and detected the expression of sLex and L-selectin in embryonic cells (jar cells) and endometrial cells (RL95-2 cells) by immunoassay to show the role of L-selectin in embryonic adhesion. The results were as follows: the correct rate of fetal sex determination by ultrasound imaging increased with the increase of pregnancy days and reached 93% at 84 days; sLex/L-selectin on the surface of Jar/RL95-2 cells is involved in the adhesion between embryo and endometrium; and when the concentration of L-selectin was 30 µg/ml, the implantation success rate of fertilized eggs and embryos was the highest, reaching 95%. It is proved that ultrasonic intelligent imaging exploration can summarize the imaging characteristics of the early development law of sheep fetus, which provides a basis for B-ultrasound to monitor fetal growth and predict fetal age. While discussing the molecular mechanism of implantation, it provides a new idea and means for the clinical intervention of contraception and pregnancy assistance with oligosaccharide as the target.