Spliceosomal genes in the D. discoideum genome: a comparison with those in H. sapiens, D. melanogaster, A. thaliana and S. cerevisiae

Little is known about pre-mRNA splicing in Dictyostelium discoideum although its genome has been completely sequenced. Our analysis suggests that pre-mRNA splicing plays an important role in D. discoideum gene expression as two thirds of its genes contain at least one intron. Ongoing curation of the genome to date has revealed 40 genes in D. discoideum with clear evidence of alternative splicing, supporting the existence of alternative splicing in this unicellular organism. We identified 160 candidate U2-type spliceosomal proteins and related factors in D. discoideum based on 264 known human genes involved in splicing. Spliceosomal small ribonucleoproteins (snRNPs), PRP19 complex proteins and late-acting proteins are highly conserved in D. discoideum and throughout the metazoa. In non-snRNP and hnRNP families, D. discoideum orthologs are closer to those in A. thaliana, D. melanogaster and H. sapiens than to their counterparts in S. cerevisiae. Several splicing regulators, including SR proteins and CUG-binding proteins, were found in D. discoideum, but not in yeast. Our comprehensive catalog of spliceosomal proteins provides useful information for future studies of splicing in D.βdiscoideum where the efficient genetic and biochemical manipulation will also further our general understanding of pre-mRNA splicing.     

Targeted deletion of mouse Rad1 leads to deficient cellular DNA damage responses

The Rad1 gene is evolutionarily conserved from yeast to human. The fission yeast Schizosaccharomyces pombeRad1 ortholog promotes cell survival against DNA damage and is required for G₂/M checkpoint activation. In this study, mouse embryonic stem (ES) cells with a targeted deletion of Mrad1, the mouse ortholog of this gene, were created to evaluate its function in mammalian cells. Mrad1^-/- ES cells were highly sensitive to ultraviolet-light (UV light), hydroxyurea (HU) and gamma rays, and were defective in G₂/M as well as S/M checkpoints. These data indicated that Mrad1 is required for repairing DNA lesions induced by UV-light, HU and gamma rays, and for mediating G₂/M and S/M checkpoint controls. We further demonstrated that Mrad1 plays an important role in homologous recombination repair (HRR) in ES cells, but a minor HRR role in differentiated mouse cells.     

Unexpected Role of the Steroid-Deficiency Protein Ecdysoneless in Pre-mRNA Splicing

The steroid hormone ecdysone coordinates insect growth and development, directing the major postembryonic transition of forms, metamorphosis. The steroid-deficient ecdysoneless¹ (ecd¹) strain of Drosophila melanogaster has long served to assess the impact of ecdysone on gene regulation, morphogenesis, or reproduction. However, ecd also exerts cell-autonomous effects independently of the hormone, and mammalian Ecd homologs have been implicated in cell cycle regulation and cancer. Why the Drosophila ecd¹ mutants lack ecdysone has not been resolved. Here, we show that in Drosophila cells, Ecd directly interacts with core components of the U5 snRNP spliceosomal complex, including the conserved Prp8 protein. In accord with a function in pre-mRNA splicing, Ecd and Prp8 are cell-autonomously required for survival of proliferating cells within the larval imaginal discs. In the steroidogenic prothoracic gland, loss of Ecd or Prp8 prevents splicing of a large intron from CYP307A2/spookier (spok) pre-mRNA, thus eliminating this essential ecdysone-biosynthetic enzyme and blocking the entry to metamorphosis. Human Ecd (hEcd) can substitute for its missing fly ortholog. When expressed in the Ecd-deficient prothoracic gland, hEcd re-establishes spok pre-mRNA splicing and protein expression, restoring ecdysone synthesis and normal development. Our work identifies Ecd as a novel pre-mRNA splicing factor whose function has been conserved in its human counterpart. Whether the role of mammalian Ecd in cancer involves pre-mRNA splicing remains to be discovered.     

Phosphorylation-dephosphorylation differentially affects activities of splicing factor ASF/SF2.

SR proteins are a conserved family of splicing factors that function in both constitutive and activated splicing. We reported previously that phosphorylation of the SR protein ASF/SF2 enhances its interaction with the U1 snRNP-specific 70K protein and is required for the protein to function in splicing, while other studies have provided evidence that subsequent dephosphorylation can also be required for SR protein function, at least in constitutive splicing. We now show that the phosphorylation status of ASF/SF2 can differentially affect several properties of the protein. In keeping with a dynamic cycle of phosphorylation-dephosphorylation during splicing, ASF/SF2 phosphorylation was found to affect interaction with several putative protein targets in different ways: positively, negatively or not at all. Extending these results, we also show that, in contrast to constitutive splicing, dephosphorylation is not required for ASF/SF2 to function as a splicing activator. We discuss these results with respect to the differential protein-protein interactions that must occur during constitutive and activated splicing.     

Crooked neck is a component of the human spliceosome and implicated in the splicing process

The Drosophila crooked neck (crn) gene is essential for embryogenesis and has been implicated in cell cycle progression and in pre-mRNA splicing although a direct role in either process has not been established. Here we report isolation of the human crooked neck homolog, HCRN, and provide evidence for its function in splicing. HCRN encodes an unusual protein composed largely of tetratricopeptide repeat (TPR) elements. The crooked neck protein co-localizes with the SR and Sm protein splicing factors in discrete subnuclear domains implicated in snRNP biogenesis. In vitro assembly experiments show that an 83 kDa hcrn isoform is stably recruited to splicing complexes coincident with the addition of the U4/U6.U5 tri-snRNP particle. Crooked neck activity appears essential as extracts depleted of hcrn fail to splice pre-mRNA. These and related data support the view that crooked neck is a phylogenetically conserved pre-mRNA splicing factor.     

KSRP suppresses cell invasion and metastasis through miR-23a-mediated EGR3 mRNA degradation in non-small cell lung cancer

KH-type splicing regulatory protein (KSRP) is a single-strand RNA binding protein which regulates mRNA stability either by binding to AU-rich elements (AREs) of mRNA 3′UTR or by facilitating miRNA biogenesis to target mRNA. Unlike its well-characterized function at the molecular level in maintaining RNA homeostasis, the role of KSRP in cancer progression remains largely unknown. Here we investigate the role of KSRP in non-small cell lung cancer (NSCLC). We first examined KSRP expression by immunohistochemistry in a cohort containing 196 NSCLC patients and observed a strong positive correlation between KSRP expression and survival of NSCLC patients. Multivariate analysis further identified KSRP as an independent prognostic factor. Manipulating KSRP expression significantly affected in vitro cell mobility and in vivo metastatic ability of NSCLC cells. Microarray analysis identified an ARE-containing gene, EGR3, as a downstream effector of KSRP in NSCLC. Interestingly, we found that KSRP decreased EGR3 mRNA stability in an ARE-independent manner. By screening KSRP-regulated miRNAs in NSCLC cells, we further found that miR-23a directly binds to EGR3 3′UTR, reducing EGR3 expression and thereby inhibiting NSCLC cell mobility. Our findings implicate a targetable KSRP/miR-23a/EGR3 signaling axis in advanced tumor phenotypes.     

Cell Cycle Abnormalities Associated with Differential Perturbations of the Human U5 snRNP Associated U5-200kD RNA Helicase

Splicing of pre-messenger RNAs into functional messages requires a concerted assembly of proteins and small RNAs that identify the splice junctions and facilitate cleavage of exon-intron boundaries and ligation of exons. One of the key steps in the splicing reaction is the recruitment of a tri-snRNP harboring the U5/U4/U6 snRNPs. The U5 snRNP is also required for both steps of splicing and exon-exon joining. One of the key components of the tri-snRNP is the U5 200kd helicase. The human U5-200kD gene isolated from Hela cells encodes a 200 kDa protein with putative RNA helicase function. Surprisingly, little is known about the functional role of this protein in humans. Therefore, we have investigated the role of the U5-200kD RNA helicase in mammalian cell culture. We created and expressed a dominant negative domain I mutant of the RNA helicase in HEK293 cells and used RNAi to downregulate expression of the endogenous protein. Transient and stable expression of the domain I mutant U5-200kD protein using an ecdysone-inducible system and transient expression of an anti-U5-200kD short hairpin RNA (shRNA) resulted in differential splicing and growth defects in the 293/EcR cells. Cell cycle analysis of the dominant negative clones revealed delayed exit from the G2/M phase of the cell cycle due to a mild splicing defect. In contrast to the domain I dominant negative mutant expressing cells, transient expression of an anti-U5-200kD shRNA resulted in a pronounced S phase arrest and a minute splicing defect. Collectively, this work demonstrates for the first time establishment of differential human cell culture splicing and cell cycle defect models due to perturbed levels of an essential core splicing factor.     

The centrosomal kinase NEK2 is a novel splicing factor kinase involved in cell survival

NEK2 is a serine/threonine kinase that promotes centrosome splitting and ensures correct chromosome segregation during the G2/M phase of the cell cycle, through phosphorylation of specific substrates. Aberrant expression and activity of NEK2 in cancer cells lead to dysregulation of the centrosome cycle and aneuploidy. Thus, a tight regulation of NEK2 function is needed during cell cycle progression. In this study, we found that NEK2 localizes in the nucleus of cancer cells derived from several tissues. In particular, NEK2 co-localizes in splicing speckles with SRSF1 and SRSF2. Moreover, NEK2 interacts with several splicing factors and phosphorylates some of them, including the oncogenic SRSF1 protein. Overexpression of NEK2 induces phosphorylation of endogenous SR proteins and affects the splicing activity of SRSF1 toward reporter minigenes and endogenous targets, independently of SRPK1. Conversely, knockdown of NEK2, like that of SRSF1, induces expression of pro-apoptotic variants from SRSF1-target genes and sensitizes cells to apoptosis. Our results identify NEK2 as a novel splicing factor kinase and suggest that part of its oncogenic activity may be ascribed to its ability to modulate alternative splicing, a key step in gene expression regulation that is frequently altered in cancer cells.     

Human transformer 2beta and SRp55 interact with a calcitonin-specific splice enhancer

The calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA is alternatively processed in a tissue-specific manner leading to the production of calcitonin mRNA in thyroid C cells and CGRP mRNA in neurons. Sequences in the human calcitonin-specific fourth exon function as an exonic splice enhancer (ESE) which is required for incorporation of exon 4 into calcitonin mRNA. Deletion of these sequences from the rat calcitonin/CGRP gene was reported to have no effect on calcitonin splicing. We demonstrate that sequences in the rat calcitonin/CGRP fourth exon act as an ESE. In addition, we observed that three proteins in HeLa nuclear extract, of apparent molecular weights of 40, 55 and 85 kDa, specifically interact with the exon 4 ESE. The 40-kDa protein is human transformer 2beta (hTra2beta), a homolog of the Drosophila splice regulator transformer 2. hTra2beta is required for calcitonin splicing in vitro, one of the first biological functions identified for hTra2beta. The 55-kDa protein is SRp55, a member of the SR family of phosphoproteins. Binding of SRp55 to an ESE required for calcitonin mRNA splicing suggests that the different levels of SRp55 present in different cell types may regulate calcitonin/CGRP alternative splicing.     

Central role for the XRCC1 BRCT I domain in mammalian DNA single-strand break repair

The DNA single-strand break repair (SSBR) protein XRCC1 is required for genetic stability and for embryonic viability. XRCC1 possesses two BRCA1 carboxyl-terminal (BRCT) protein interaction domains, denoted BRCT I and II. BRCT II is required for SSBR during G(1) but is dispensable for this process during S/G(2) and consequently for cell survival following DNA alkylation. Little is known about BRCT I, but this domain has attracted considerable interest because it is the site of a genetic polymorphism that epidemiological studies have associated with altered cancer risk. We report that the BRCT I domain comprises the evolutionarily conserved core of XRCC1 and that this domain is required for efficient SSBR during both G(1) and S/G(2) cell cycle phases and for cell survival following treatment with methyl methanesulfonate. However, the naturally occurring human polymorphism in BRCT I supported XRCC1-dependent SSBR and cell survival after DNA alkylation equally well. We conclude that while the BRCT I domain is critical for XRCC1 to maintain genetic integrity and cell survival, the polymorphism does not impact significantly on this function and therefore is unlikely to impact significantly on susceptibility to cancer.