Abstract
In eukaryotic cells, RNAs transcribed by RNA polymerase-II receive the modification at the 5′ end. This structure is called the cap structure. The cap structure has a fundamental role for ...translation initiation by recruiting eukaryotic translation initiation factor 4F (eIF4F). The other important mediator of the cap structure is a nuclear cap-binding protein complex (CBC). CBC consists of two proteins, which are renamed as NCBP1 and NCBP2 (previously called as CBP80/NCBP and CBP20/NIP1, respectively). This review article discusses the multiple roles CBC mediates and co-ordinates in several gene expression steps in eukaryotes.
Graphical Abstract
Graphical Abstract
Pre-mRNA splicing is an essential step for gene expression in higher eukaryotes. Alternative splicing contributes to diversity of the expressed proteins from the limited number of genes. Disruption ...of splicing regulation often results in hereditary and sporadic diseases called as ‘RNA diseases’. Modulation of splicing by small chemical compounds and nucleic acids has been tried to target aberrant splicing in those diseases. Several RNA diseases and splicing-target therapeutic approaches will be briefly introduced in this review. Accumulating knowledge about molecular mechanism of aberrant splicing and their correction by chemical compounds is important not only for RNA biologists, but also for clinicians who desire therapies for those diseases.
Eukaryotes are often subjected to different kinds of stress. In order to adjust to such circumstances, eukaryotes activate stress-response pathways and regulate gene expression. Eukaryotic gene ...expression consists of many different steps, including transcription, RNA processing, RNA transport, and translation. In this review article, we focus on both transcriptional and post-transcriptional regulations of gene expression under hypoxic conditions. In the first part of the review, transcriptional regulations mediated by various transcription factors including Hypoxia-Inducible Factors (HIFs) are described. In the second part, we present RNA splicing regulations under hypoxic conditions, which are mediated by splicing factors and their kinases. This work summarizes and discusses the emerging studies of those two gene expression machineries under hypoxic conditions.
In eukaryotes, pre-mRNA splicing is an essential step for gene expression. Splicing reactions have been well investigated by using in vitro splicing reactions with extracts prepared from cultured ...cells. Here, we describe protocols for the preparation of splicing-competent extracts from cells expressing a tagged spliceosomal protein. The whole-cell extracts are able to splice exogenously added pre-mRNA and the RNA-protein complex formed in the in vitro splicing reaction can be purified by immunoprecipitation using antibodies against the peptide tag on the splicing protein. The method described here to prepare splicing-active extracts from whole cells is particularly useful when studying pre-mRNA splicing in various cell types, and the expression of a tagged spliceosomal protein allows one to purify and analyze the RNA-protein complexes by simple immunoprecipitation.
It has been assumed that premessenger ribonucleic acids (RNAs; pre-mRNAs) are spliced cotranscriptionally in the process of gene expression. However, in this paper, we report that splicing of Clk1/4 ...mRNAs is suspended in tissues and cultured cells and that intermediate forms retaining specific introns are abundantly pooled in the nucleus. Administration of the Cdc2-like kinase-specific inhibitor TG003 increased the level of Clk1/4 mature mRNAs by promoting splicing of the intron-retaining RNAs. Under stress conditions, splicing of general pre-mRNAs was inhibited by dephosphorylation of SR splicing factors, but exposure to stresses, such as heat shock and osmotic stress, promoted the maturation of Clk1/4 mRNAs. Clk1/4 proteins translated after heat shock catalyzed rephosphorylation of SR proteins, especially SRSF4 and SRSF10. These findings suggest that Clk1/4 expression induced by stress-responsive splicing serves to maintain the phosphorylation state of SR proteins.
Pre-mRNA splicing is an essential process for gene expression in higher eukaryotes, which requires a high order of accuracy. Mutations in splicing factors or regulatory elements in pre-mRNAs often ...result in many human diseases. Myelodysplastic syndrome (MDS) is a heterogeneous group of chronic myeloid neoplasms characterized by many symptoms and a high risk of progression to acute myeloid leukemia. Recent findings indicate that mutations in splicing factors represent a novel class of driver mutations in human cancers and affect about 50% of Myelodysplastic syndrome (MDS) patients. Somatic mutations in MDS patients are frequently found in genes SF3B1, SRSF2, U2AF1, and ZRSR2. Interestingly, they are involved in the recognition of 3′ splice sites and exons. It has been reported that mutations in these splicing regulators result in aberrant splicing of many genes. In this review article, we first describe molecular mechanism of pre-mRNA splicing as an introduction and mainly focus on those four splicing factors to describe their mutations and their associated aberrant splicing patterns.
Alternative splicing of the pyruvate kinase M (PKM) pre-mRNA generates two isoforms, PKM1 and PKM2. PKM catalyzes the conversion of phosphoenol-pyruvate to pyruvate in glycolytic pathway. PKM1 exist ...as a stable tetramer that is at an active enzyme state, while PKM2 is in equilibrium among monomer, dimer and tetramer under the regulation of its allosteric activators. Many cancer cells show the feature of higher glucose uptake and lactate production in spite of oxygen availability, which is known as the Warburg effect. PKM2 is upregulated in most cancer types and the inactive PKM2 lead to the cancer metabolism. In addition, dimeric PKM2 induces its nuclear translocation through posttranslational modification and acts as a transcriptional co-activator for the expression of oncogenes. Therefore, it is important to elucidate mechanisms for modulation of an active or inactive state of PKM2, namely the tetramer-to-dimer-transition. The definitive difference between PKM1 and PKM2 is to constitutively form tetramer or not in the cytoplasm, which is ascribed to 22 amino acids derived from exon 9 (PKM1) or exon 10 (PKM2). In this study, we generated 22 different PKM1-mimetic point mutants of PKM2, and demonstrated that replacement of cysteine424 residue of PKM2 with leucine424 conserved in PKM1 (C424L) promote its tetramerization. PKM2(C424L) formed a tetramer without allosteric activator, and escaped the inhibitory effects by oxidative stress, like PKM1. Our findings intensely suggest that C424 or L424 determines the different catalytic and modulatory properties between PKM splicing isoforms.
•The major difference between PKM1 and PKM2 is to form a stable tetramer or not.•Replacement of the cysteine with leucine at 424th amino acid residue in PKM2 promotes tetramerization as PKM1.•The C424 residue is responsible for the characteristics of PKM2 in susceptibility against oxidative stress.
SRSF4 is one of the members of serine‐/arginine (SR)‐rich protein family involved in both constitutive and alternative splicing. SRSF4 is localized in the nucleus with speckled pattern, but its ...nuclear localization signal was not determined. Here, we have identified nuclear localization signals (NLSs) of SRSF4 by using a pyruvate kinase fusion system. As expected, arginine‐/serine (RS)‐rich domain of SRSF4 confers nuclear localization activity when it is fused to PK protein. We then further delineated the minimum sequences for nuclear localization in RS domain of SRSF4. Surprisingly, RS‐rich region does not always have a nuclear localization activity. In addition, basic amino acid stretches that resemble to classical‐type NLSs were identified. These results strongly suggest that SRSF4 protein uses two different nuclear import pathways with multiple NLSs in RS domain.
SRSF4 is a splicing factor that belongs to serine‐/arginine‐rich (SR) protein splicing factor family. By using reporter protein system, we have identified nuclear localization signals (NLSs) of SRSF4. It was assumed that arginine‐/serine‐rich (RS) domain of SRSF4 confers its nuclear localization activity. However, we found that SRSF4 has not only RS repeat‐type NLSs but also classical‐type NLSs. These results strongly suggest that SRSF4 protein uses two different nuclear import pathways with multiple NLSs in RS domain.
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