LINC00261 regulates EBF1 to suppress malignant progression of thyroid cancer.

OBJECTIVE: We aimed to explore the role of LINC00261 in thyroid cancer (TC) and the potential regulatory mechanism. PATIENTS AND METHODS: 40 cases of tumor tissues and adjacent tissues of TC patients were collected, and the expressions of LINC00261 and EBF1 were detected by quantitative real-time polymerase chain reaction (qRT-PCR), and the relationship between LINC00261 and the clinical pathological indicators and prognosis of TC patients were analyzed. Next, LINC00261 overexpression and knockdown cell models were constructed in TC cell lines BPH5-16 and K1, respectively. Cell counting kit-8 (CCK-8) and transwell migration were used to detect the impact of LINC00261 overexpression or silencing on cell proliferative and migration ability. The bioinformatics website was used to screen the possible target gene of LINC00261. RESULTS: qRT-PCR analysis showed that LINC00261 level was markedly reduced in TC tumor tissues, as well as corresponding cell lines. Retrospective analysis showed that low expression of LINC00261 was in positive correlation with the pathological stage, lymphatic and distant metastasis in patients with TC, meanwhile, the expression of LINC00261 was also in positive correlation with overall survival rate of TC patients. Bioinformatics analysis suggested that LINC00261 could target EBF1. Luciferase reporter gene experiment and qRT-PCR analysis suggested that LINC00261 could target EBF1 and that their expressions showed a negative correlation in TC tumor tissues and cells. Cell functional experiments confirmed that LINC00261 can inhibit the proliferative and migration ability of TC cells. Subsequently, the recovery experiment also suggested that silencing EBF1 could reverse the promotion effect of LINC00261 knockdown on the proliferative and migration ability of TC cells; while EBF1 overexpression could reverse the inhibition of LINC00261 on the proliferative and migration ability of TC cells. CONCLUSIONS: LINC00261 was markedly downregulated in TC tissues and cells. In addition, the level of LINC00261 was closely related to lymph node and distant metastasis, as well as the prognosis in TC patients. Moreover, LINC00261 could negatively regulate EBF1, thereby promoting the malignant progression of TC.

Ursodeoxycholic acid and S-adenosylmethionine in the treatment of intrahepatic cholestasis of pregnancy: a multi-centered randomized controlled trial.

OBJECTIVE: Intrahepatic cholestasis of pregnancy (ICP) is a special complication of pregnancy characterized by skin pruritus, abnormal liver function tests and bile acids. To compare the efficacy of ursodeoxycholic acid (UDCA) and S-adenosylmethionine (SAMe) monotherapy with their combined effect on intrahepatic cholestasis of pregnancy (ICP). PATIENTS AND METHODS: Singleton pregnancies with ICP in five tertiary medical centers were randomly divided into three treatment groups: oral UDCA 4×250 mg daily (Group 1, n = 41), intravenous SAMe 1000 mg daily (Group 2, n = 38), and a combination of both drugs (Group 3, n = 41) until delivery. Paired t test, analysis of covariance and non-parametric test were used. RESULTS: All therapies significantly and equally improved pruritus. The serum levels of total bile acids (TBA), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TB) in each group significantly decreased after treatment (p < 0.05). Group 1 was more effective than Group 2 in reducing TBA concentration (p < 0.05), Group 1 and Group 3 showed more effective than Group 2 in reducing AST and TB concentrations (p < 0.05), and Group 1 facilitated deliveries at term. No perinatal death or adverse drug reactions were observed. CONCLUSIONS: UDCA and SAMe are both effective and safe in the treatment of ICP. UDCA monotherapy should be used as the first line therapy for ICP because it is more efficacious, cost-effective and convenient.

Effect of hypothalamic-pituitary-adrenal axis alterations on glucose and lipid metabolism in diabetic rats.

This study investigated the relationship between alterations in the hypothalamic-pituitary-adrenal (HPA) axis function and glucose and lipid metabolism in diabetic rats. To accomplish this a diabetes model was established by jointly administering a long-term high-fat diet plus Streptozotocin (STZ; 50 mg/kg ip). The rats were randomly divided into four groups: 1) a normal control group, 2) a model group, 3) astragalus polysaccharide (APS) group, and 4) a metformin group. APS and metformin hydrochloride were administered intragastrically (100 mg∙kg(-1)d(-1)). Rat blood glucose and body weight were measured once per week, and urine was collected for 24 h after 30 days of administration of APS. The levels of blood lipids, insulin, and corticosterone (CORT), as well as hypothalamic CRH, pituitary ACTH, urine sugar and CORT were measured. Compared with the normal control group, the levels of blood sugar, urine sugar, TC, and TG significantly increased in the model group, and the levels of hepatic glycogen and HDL-C decreased. Administration of APS was shown to reverse these changes. Furthermore, as compared with the normal control group, the levels of insulin and hypothalamic CRH in the model group decreased significantly, while the levels of plasma ACTH and CORT, pituitary ACTH, and urine CORT were elevated. Again, APS administration improves these outcomes and returns their levels to normal. Thus, the glucose and lipid metabolic disorder in the high-fat diet and STZ-induced diabetes model may be related to increased HPA axis activity. The hypoglycemic effect of the traditional Chinese medicine, ASP, may improve HPA axis functioning and aid in the treatment of diabetes.

Isolation and analysis of cell-free fetal DNA from maternal peripheral blood in Chinese women.

Non-invasive prenatal diagnosis is used to detect the genetic material of the fetus by isolating the cell-free fetal DNA (cffDNA) from maternal peripheral blood. In order to establish an isolation method for cffDNA from maternal peripheral blood in Chinese women, the cffDNA was acquired with a two-step centrifugation using a QlAamp DNA Blood mini kit. The SRY gene of plasma DNA was amplified by polymerase chain reaction (PCR). Real-time quantitative PCR was used to measure the concentration of cffDNA in maternal peripheral blood in different pregnant women. The results of the SRY gene amplification of plasma DNA from pregnant women was the same as that of the amniocyte DNA. The average concentration of cffDNA in maternal peripheral blood of pregnant women in different gestational stages was 0.98 ng/mL (0.26-1.49 ng/mL), 1.43 ng/mL (0.46- 2.34 ng/mL), and 1.95 ng/mL (0.65-6.81 ng/mL) from early, middle, and late gestational stages, respectively. The mean of cffDNA from total DNA in plasma in different stages of gestation was 22.28% (9.86-27.81%). The lowest concentration of DNA amplified by nested-PCR in our research was 10-4-10-3 ng/µL. The isolation method for cffDNA from maternal peripheral blood was successfully established and further research into its applications will be conducted.

Preparation and biological activity of a paclitaxel-single-walled carbon nanotube complex.

Single-walled carbon nanotubes (SWCNTs) have unique transmembrane abilities. The huge superficial area and abundance of π electrons confer SWCNTs perfect absorptive capability toward proteins, nucleates, and many drugs. These characteristics make SWCNTs a new and efficient drug carrier. The purpose of this study was to disperse SWCNTs in water and have paclitaxel absorbed onto them in order to construct an asparagine-glycine-arginine (NGR)-SWCNT-Paclitaxel complex as a targeting nanoparticle system. The NGR-SWCNT-Paclitaxel complex was systematically studied, and analytical methods, including spectrophotometry for SWCNTs and high-performance liquid chromatography for paclitaxel, were employed. The preparation and the prescription of the NGR-SWCNT-Paclitaxel complex lyophilized powder were investigated. MCF-7 cancer cells, Sprague-Dawley rats, and S180 tumor-bearing mice were used as experimental subjects to evaluate the in vitro and in vivo activity of NGR-SWCNT-Paclitaxel complex dispersion. The complex dispersion showed obvious inhibition activity against MCF-7 cancer cells. Within 1 h, the NGR-SWCNT-Paclitaxel complex could be transferred to cells, and sustained the release of drugs. In addition, the tumor and liver targeting and improved therapeutic effects of the NGR-SWCNT-Paclitaxel complex were confirmed.

Genetic polymorphisms of the TLR4 gene and their association with susceptibility to type 2 diabetes mellitus in the Chinese population.

Toll-like receptor 4 (TLR4) is potentially an important gene affecting the susceptibility to type 2 diabetes mellitus (T2DM). The objective of this study was to evaluate whether genetic polymorphisms of the TLR4 gene are associated with T2DM susceptibility. This potential association was analyzed in 668 T2DM patients and 672 healthy controls by polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing methods. Two novel genetic polymorphisms (g.12375A>G and g.14367G>A) were investigated, and our data support the idea that the g.14367G>A variant significantly increased susceptibility to T2DM in homozygote comparison (AA vs GG: OR = 2.396, 95%CI = 1.682-3.413, P < 0.0001), heterozygote comparison (GA vs AA: OR = 1.322, 95%CI = 1.050-1.664, P = 0.0175), dominant model (AA/GA vs GG: OR = 1.511, 95%CI = 1.217-1.876, P = 0.0002), recessive model (AA vs GA/GG: OR = 2.093, 95%CI = 1.496-2.927, P < 0.0001), and allele contrast (A vs G: OR = 1.503, 95%CI = 1.279-1.766, P < 0.0001). The allele A of g.14367G>A variants may contribute to the susceptibility to T2DM. However, we failed to detect a similar significantly increased susceptibility to T2DM in the g.12375A>G variant. Our findings suggest that the g.14367G>A genetic polymorphism of the TLR4 gene is associated with the susceptibility to T2DM in the population studied.

Endothelial regulation of eNOS, PAI-1 and t-PA by testosterone and dihydrotestosterone in vitro and in vivo.

The aim of this study is the identification of direct endothelial regulation by the androgens testosterone (T) and dihydrotestosterone (DHT). We tested the effects of T and DHT on nitric oxide (NO) synthesis and on tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) expression in human endothelial cells and in ovariectomized (OVX) rats. The results showed that at physiological concentrations T and DHT increase endothelial synthesis of NO. This depends on a rapid recruitment of the extracellular-related kinase (ERK) 1/2 and of the phosphatidylinositol 3-OH kinase (PI3K)/Akt cascades, resulting in endothelial nitric oxide synthase (eNOS) Ser(1177)-phosphorylation. In addition, a later increase of eNOS expression is found. With supra-physiological amounts of T or DHT the induction of NO synthesis is lost. A concentration-related increase of t-PA expression starting from physiological concentrations of T or DHT is found, whereas PAI-1 is augmented only with higher doses. Although DHT exerts these actions through androgen receptors (AR), T acts in part through aromatase-dependent conversion to 17ß-estradiol. Ovariectomy is associated with significant changes in eNOS, t-PA and PAI-1 expression in the aorta of Wistar rats and T and DHT result in modifications on eNOS, PAI-1 and t-PA that are in line with the in vitro experiments. In conclusion, T and DHT act on endothelial cells through AR or via conversion to estradiol. Physiological, but not higher amounts are associated with enhanced NO synthesis and an increased t-PA/PAI-1 ratio. These findings are useful to understand the impact of androgens in ageing individuals.

Drospirenone increases endothelial nitric oxide synthesis via a combined action on progesterone and mineralocorticoid receptors.

BACKGROUND: Progestins have actions on the cardiovascular system, which depend on the structure as well as on receptor binding characteristics. Drospirenone (DRSP) is a progestin that uniquely interferes with the signaling of the mineralocorticoid receptor (MR). Hormone therapy containing DRSP results in blood pressure reduction in hypertensive post-menopausal women. METHODS: We describe the effects of DRSP on endothelial nitric oxide (NO) synthesis and compare them with those of progesterone (P) and of medroxyprogesterone acetate (MPA). In addition, we herein tested the relevance of the anti-mineralocorticoid activity of DRSP for NO synthesis. RESULTS: DRSP results in rapid activation of the endothelial NO synthase (eNOS) through mitogen-activated protein kinases and phosphatidylinositol 3-kinase as well as in enhanced eNOS expression. These actions depend on P receptor. When the cells are exposed to aldosterone, a reduction of eNOS expression is found that is antagonized by DRSP. This action is not shared by P or MPA. In addition, DRSP does not interfere with the induction or activation of eNOS induced by estradiol, as opposed to MPA. CONCLUSIONS: DRSP acts on endothelial cells via a combined action through the P and MRs. These results help to interpret the anti-hypertensive effects of hormonal therapies containing DRSP.

A database designed to computationally aid an experimental approach to alternative splicing.

A unique microarray approach has been developed to profile alternative splicing in the cell. To support the development of this approach, we have developed the Manually Annotated Alternatively Spliced Events (MAASE) database system, which is a unique alternative splicing information resource designed specifically with experimentalists in mind. MAASE is an online resource for the convenient access, identification, and annotation of alternative splicing events (ASEs). MAASE consists of two components: an annotation system and a curated database. The annotation system is a web-based workspace that combines manual and computational approaches to identifying and annotating ASEs, a combination that is vital if a comprehensive collection is to be obtained. The annotation system is publicly available and provides a scalable solution to acquiring as well as contributing to annotated ASEs. MAASE annotated ASEs are deposited into the database component, which can either be queried one entry at a time or multiple entries at a time with convenient access to alternatively spliced junctional and surrounding sequences to facilitate the design of microarray experiments.

Evidence for a role of Sky1p-mediated phosphorylation in 3' splice site recognition involving both Prp8 and Prp17/Slu4.

The SRPK family of kinases is specific for RS domain-containing splicing factors and known to play a critical role in protein-protein interaction and intracellular distribution of their substrates in both yeast and mammalian cells. However, the function of these kinases in pre-mRNA splicing remains unclear. Here we report that SKY1, a SRPK family member in Saccharomyces cerevisiae, genetically interacts with PRP8 and PRP17/SLU4, both of which are involved in splice site selection during pre-mRNA splicing. Prp8 is essential for splicing and is known to interact with both 5' and 3' splice sites in the spliceosomal catalytic center, whereas Prp17/Slu4 is nonessential and is required only for efficient recognition of the 3' splice site. Interestingly, deletion of SKY1 was synthetically lethal with all prp17 mutants tested, but only with specific prp8 alleles in a domain implicated in governing fidelity of 3'AG recognition. Indeed, deletion of SKY1 specifically suppressed 3'AG mutations in ACT1-CUP1 splicing reporters. These results suggest for the first time that 3' AG recognition may be subject to phosphorylation regulation by Sky1p during pre-mRNA splicing.

SC35 plays a role in T cell development and alternative splicing of CD45.

Molecular diversity via alternative splicing is important for cellular function and development. SR proteins are strong candidate regulators of alternative splicing because they can modulate splice site selection. However, endogenous substrates for SR proteins are largely unknown, and their roles as splicing regulators in vertebrate development are unclear. Here we report that Cre-mediated conditional deletion of the prototypical SR protein SC35 in the thymus causes a defect in T cell maturation. Deletion of SC35 alters alternative splicing of CD45, a receptor tyrosine phosphatase known to be regulated by differential splicing during thymocyte development and activation. This study establishes a model to address the function of SR proteins in physiological settings and reveals a critical role of SC35 in a T cell-specific regulated splicing pathway.

The structure of Sky1p reveals a novel mechanism for constitutive activity.

Sky1p is the only member of the SR protein kinase (SRPK) family in Saccharomyces cerevisiae. SRPKs are constitutively active kinases that display remarkable substrate specificity and have been implicated in RNA processing. Here we present the three-dimensional structure of a fully active truncated Sky1p. Analysis of the structure and structure-based functional studies reveal that the C-terminal tail, an unusual Glu residue located in the P+1 loop, and a unique mechanism for the positioning of helix alpha C act together to render Sky1p constitutively active. We have modeled a substrate peptide bound to Sky1p. The modeled complex combined with mutagenesis studies illustrate the molecular basis for substrate recognition by this kinase and suggest a mechanism by which SRPKs catalyze a sequential phosphorylation reaction of the consecutive RS dipeptide repeats characteristic of mammalian SRPK substrates.

Conserved SR protein kinase functions in nuclear import and its action is counteracted by arginine methylation in Saccharomyces cerevisiae.

Mammalian serine and arginine-rich (SR) proteins play important roles in both constitutive and regulated splicing, and SR protein-specific kinases (SRPKs) are conserved from humans to yeast. Here, we demonstrate a novel function of the single conserved SR protein kinase Sky1p in nuclear import in budding yeast. The yeast SR-like protein Npl3p is known to enter the nucleus through a composite nuclear localization signal (NLS) consisting of a repetitive arginine- glycine-glycine (RGG) motif and a nonrepetitive sequence. We found that the latter is the site for phosphorylation by Sky1p and that this phosphorylation regulates nuclear import of Npl3p by modulating the interaction of the RGG motif with its nuclear import receptor Mtr10p. The RGG motif is also methylated on arginine residues, but methylation does not affect the Npl3p-Mtr10p interaction in vitro. Remarkably, arginine methylation interferes with Sky1p-mediated phosphorylation, thereby indirectly influencing the Npl3p-Mtr10p interaction in vivo and negatively regulating nuclear import of Npl3p. These results suggest that nuclear import of Npl3p is coordinately influenced by methylation and phosphorylation in budding yeast, which may represent conserved components in the dynamic regulation of RNA processing in higher eukaryotic cells.

Conservation in budding yeast of a kinase specific for SR splicing factors.

SR protein kinases (SRPKs) and their substrates, the SR family of serine/arginine-rich pre-mRNA splicing factors, appear to be key regulators of alternative splicing. Although SR proteins have been well characterized through biochemical experiments in metazoans, their functions in vivo are unclear. Because of the strict splice site consensus and near absence of alternative splicing in Saccharomyces cerevisiae, it had been thought that budding yeast would lack an SRPK and its substrates. Here, we present structural, biochemical, and cell-biological evidence that directly demonstrates an SR protein kinase, Sky1p, as well as a number of SRPK substrates in S. cerevisiae. One of these substrates is Npl3p, an SR-like protein involved in mRNA export. This finding raises the provocative possibility that Sky1p, and by extension metazoan SRPKs, regulates mRNA export or the nucleocytoplasmic shuttling of RS domain proteins. The unexpected discovery of an SR protein kinase in budding yeast provides a foundation for genetic dissection of the biological functions of SR proteins and their kinases.

Phosphorylation regulates in vivo interaction and molecular targeting of serine/arginine-rich pre-mRNA splicing factors.

The SR superfamily of splicing factors and regulators is characterized by arginine/serine (RS)-rich domains, which are extensively modified by phosphorylation in cells. In vitro binding studies revealed that RS domain-mediated protein interactions can be differentially affected by phosphorylation. Taking advantage of the single nonessential SR protein-specific kinase Sky1p in Saccharomyces cerevisiae, we investigated RS domain interactions in vivo using the two-hybrid assay. Strikingly, all RS domain-mediated interactions were abolished by SKY1 deletion and were rescuable by yeast or mammalian SR protein-specific kinases, indicating that phosphorylation has a far greater impact on RS domain interactions in vivo than in vitro. To understand this dramatic effect, we examined the localization of SR proteins and found that SC35 was shifted to the cytoplasm in sky1Delta yeast, although this phenomenon was not obvious with ASF/SF2, indicating that nuclear import of SR proteins may be differentially regulated by phosphorylation. Using a transcriptional repression assay, we further showed that most LexA-SR fusion proteins depend on Sky1p to efficiently recognize the LexA binding site in a reporter, suggesting that molecular targeting of RS domain-containing proteins within the nucleus was also affected. Together, these results reveal multiple phosphorylation-dependent steps for SR proteins to interact with one another efficiently and specifically, which may ultimately determine the splicing activity and specificity of these factors in mammalian cells.

Localization of serine kinases, SRPK1 (SFRSK1) and SRPK2 (SFRSK2), specific for the SR family of splicing factors in mouse and human chromosomes.

The serine- and arginine-rich (SR) splicing factors play an important role in both constitutive and alternative pre-mRNA splicing, and the functions of these splicing factors are regulated by phosphorylation. We have previously characterized SRPK1 (SFRSK1) and SRPK2 (SFRSK2), which are highly specific protein kinases for the SR family of splicing factors. Here we report the chromosomal localization of the mouse and human genes for both kinases. SRPK1 probes detected two loci that were mapped to mouse Chromosomes 17 and X using The Jackson Laboratory interspecific backcross DNA panel, and SRPK2 probes identified a single locus on mouse Chromosome 5. Using a somatic cell hybrid mapping panel and by fluorescence in situ hybridization, SRPK1 and SRPK2 were respectively mapped to human chromosomes 6p21.2-p21.3 (a region of conserved synteny to mouse Chromosome 17) and 7q22-q31.1 (a region of conserved synteny to mouse Chromosome 5). In addition, we also found multiple SRPK-related sequences on other human chromosomes, one of which appears to correspond to a SRPK2 pseudogene on human chromosome 8.

Substrate specificities of SR proteins in constitutive splicing are determined by their RNA recognition motifs and composite pre-mRNA exonic elements.

We report striking differences in the substrate specificities of two human SR proteins, SF2/ASF and SC35, in constitutive splicing. beta-Globin pre-mRNA (exons 1 and 2) is spliced indiscriminately with either SR protein. Human immunodeficiency virus tat pre-mRNA (exons 2 and 3) and immunoglobulin mu-chain (IgM) pre-mRNA (exons C3 and C4) are preferentially spliced with SF2/ASF and SC35, respectively. Using in vitro splicing with mutated or chimeric derivatives of the tat and IgM pre-mRNAs, we defined specific combinations of segments in the downstream exons, which mediate either positive or negative effects to confer SR protein specificity. A series of recombinant chimeric proteins consisting of domains of SF2/ASF and SC35 in various combinations was used to localize trans-acting domains responsible for substrate specificity. The RS domains of SF2/ASF and SC35 can be exchanged without effect on substrate specificity. The RNA recognition motifs (RRMs) of SF2/ASF are active only in the context of a two-RRM structure, and RRM2 has a dominant role in substrate specificity. In contrast, the single RRM of SC35 can function alone, but its substrate specificity can be influenced by the presence of an additional RRM. The RRMs behave as modules that, when present in different combinations, can have positive, neutral, or negative effects on splicing, depending upon the specific substrate. We conclude that SR protein-specific recognition of specific positive and negative pre-mRNA exonic elements via one or more RRMs is a crucial determinant of the substrate specificity of SR proteins in constitutive splicing.

Ultrastructural analysis of transcription and splicing in the cell nucleus after bromo-UTP microinjection.

In this study we demonstrate, at an ultrastructural level, the in situ distribution of heterogeneous nuclear RNA transcription sites after microinjection of 5-bromo-UTP (BrUTP) into the cytoplasm of living cells and subsequent postembedding immunoelectron microscopic visualization after different labeling periods. Moreover, immunocytochemical localization of several pre-mRNA transcription and processing factors has been carried out in the same cells. This high-resolution approach allowed us to reveal perichromatin regions as the most important sites of nucleoplasmic RNA transcription and the perichromatin fibrils (PFs) as in situ forms of nascent transcripts. Furthermore, we show that transcription takes place in a rather diffuse pattern, without notable local accumulation of transcription sites. RNA polymerase II, heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins, general transcription factor TFIIH, poly(A) polymerase, splicing factor SC-35, and Sm complex of small nuclear ribonucleoproteins (snRNPs) are associated with PFs. This strongly supports the idea that PFs are also sites of major pre-mRNA processing events. The absence of nascent transcripts, RNA polymerase II, poly(A) polymerase, and hnRNPs within the clusters of interchromatin granules rules out the possibility that this domain plays a role in pre-mRNA transcription and polyadenylation; however, interchromatin granule-associated zones contain RNA polymerase II, TFIIH, and Sm complex of snRNPs and, after longer periods of BrUTP incubation, also Br-labeled RNA. Their role in nuclear functions still remains enigmatic. In the nucleolus, transcription sites occur in the dense fibrillar component. Our fine structural results show that PFs represent the major nucleoplasmic structural domain involved in active pre-mRNA transcriptional and processing events.

Role of the constitutive splicing factors U2AF65 and SAP49 in suboptimal RNA splicing of novel retroviral mutants.

Retroviruses display a unique form of alternative splicing in which both spliced and unspliced RNAs accumulate in the cytoplasm. Simple retroviruses, such as avian sarcoma virus, do not encode regulatory proteins that affect splicing; this process is controlled solely through interactions between the viral RNA and the host cell splicing machinery. Previously, we described the selection and characterization of novel avian sarcoma virus mutants. These viruses were separated into two classes based upon analysis of splicing intermediates produced in infected cells and in a cell-free system. One class, which included mutants with altered polypyrimidine tract or branch point sequences, showed significant accumulation of intermediates, suggesting that splicing was regulated in step 2. The other class, which included mutants with deletions of exonic enhancer sequences, did not accumulate splicing intermediates, suggesting that splicing was regulated before step 1 of the splicing reaction. In this report, we show that a mutant blocked at step 1 fails to form a stable spliceosomal complex, whereas one blocked at step 2 shows a defect in its ability to transit through the last spliceosomal complex. Using UV cross-linking methods, we show that regulation at each step is associated with specific changes in the binding of cellular splicing factors. Regulation at step 1 is correlated with decreased cross-linking of the factor U2AF65, whereas regulation at step 2 is correlated with enhanced cross-linking of the factor SAP49. Because these mutations were isolated by selection for replication-competent viruses, we conclude that retroviral splicing may be regulated in vivo through altered binding of constitutive splicing factors.

SRPK2: a differentially expressed SR protein-specific kinase involved in mediating the interaction and localization of pre-mRNA splicing factors in mammalian cells.

Reversible phosphorylation plays an important role in pre-mRNA splicing in mammalian cells. Two kinases, SR protein-specific kinase (SRPK1) and Clk/Sty, have been shown to phosphorylate the SR family of splicing factors. We report here the cloning and characterization of SRPK2, which is highly related to SRPK1 in sequence, kinase activity, and substrate specificity. Random peptide selection for preferred phosphorylation sites revealed a stringent preference of SRPK2 for SR dipeptides, and the consensus derived may be used to predict potential phosphorylation sites in candidate arginine and serine-rich (RS) domain-containing proteins. Phosphorylation of an SR protein (ASF/SF2) by either SRPK1 or 2 enhanced its interaction with another RS domain-containing protein (U1 70K), and overexpression of either kinase induced specific redistribution of splicing factors in the nucleus. These observations likely reflect the function of the SRPK family of kinases in spliceosome assembly and in mediating the trafficking of splicing factors in mammalian cells. The biochemical and functional similarities between SRPK1 and 2, however, are in contrast to their differences in expression. SRPK1 is highly expressed in pancreas, whereas SRPK2 is highly expressed in brain, although both are coexpressed in other human tissues and in many experimental cell lines. Interestingly, SRPK2 also contains a proline-rich sequence at its NH2 terminus, and a recent study showed that this NH2-terminal sequence has the capacity to interact with a WW domain protein in vitro. Together, our studies suggest that different SRPK family members may be uniquely regulated and targeted, thereby contributing to splicing regulation in different tissues, during development, or in response to signaling.