Splicing

Agrobacterium tumefaciens is a commonly used tool for transforming dicotyledonous plants. The underlying mechanism of transformation however is not very well understood. One problem complicating the analysis of this mechanism is the fact that most indicator genes are already active in Agrobacterium, thereby preventing the precise determination of timing and localisation of T-DNA transfer to plant cells. In order to overcome this obstacle a modified prokaryotic indicator gene was constructed. The expression of this indicator gene and its use in analysing early events in Agrobacterium-mediated plant transformation are described. A portable intron, derived from a plant intron, was introduced into the β-glucuronidase (GUS) gene. In transgenic plants containing this chimaeric gene the intron is spliced efficiently, giving rise to GUS enzymatic activity. Mapping of the splice junction indicates the exact removal of the intron. No GUS activity is detected in agrobacteria containing this construct due to the lack of a eukaryotic splicing apparatus in prokaryotes. Early phases after transformation of Arabidopsis cotyledon explants were analysed using this GUS-intron chimaeric gene showing that as early as 36 h after Agrobacterium infection significant GUS activity is detected. In vivo GUS staining of transformed cells clearly shows that quickly proliferating calli expressing GUS activity are formed, mainly at the cut surface. Minor transformation events occur however throughout the whole cotyledon. These data indicate that Agrobacterium-mediated T-DNA transfer to plants is much more efficient than has been judged from experiments where selection is applied immediately. The intron-containing GUS gene can be used as an optimised marker gene in transient and stable transformation experiments.

Hu proteins are RNA-binding proteins involved in diverse biological processes. The neuronal members of the Hu family, HuB, HuC, and HuD play important roles in neuronal differentiation and plasticity, while the ubiquitously expressed family member, HuR, has numerous functions mostly related to cellular stress response. The pivotal roles of Hu proteins are dictated by their molecular functions affecting a large number of target genes. Hu proteins affect many post-transcriptional aspects of RNA metabolism, from splicing to translation. In this communication, we will focus on these molecular events and review our current understanding of how Hu proteins mediate them. In particular, emphasis will be put on the nuclear functions of these proteins, which were recently discovered. Three examples including calcitonin/calcitonin gene-related peptide, neurofibromatosis type 1, and Ikaros will be discussed in detail. In addition, an intriguing theme of antagonism between Hu proteins and other AU-rich sequence binding proteins will be discussed.

A component present in splicing extracts selectively binds the 3' splice site of a precursor messenger RNA (pre-mRNA) transcript of a human beta-globin gene. Since this component can be immunoprecipitated by either autoantibodies of the Sm class or antibodies specifically directed against trimethylguanosine, it is a small nuclear ribonucleoprotein (snRNP). Its interaction with the 3' splice site occurs rapidly even at 0 degrees C, does not require adenosine triphosphate, and is altered by certain mutations in the 3' splice site region. Binding is surprisingly insensitive to treatment of the extract with micrococcal nuclease. The U5 particle is the only abundant Sm snRNP with a capped 5' end that is equally resistant to micrococcal nuclease. This suggests that, in addition to the U1 and U2 snRNP's, U5 snRNP's participate in pre-mRNA splicing.

U2 auxiliary factor (U2AF) is a non-snRNP protein required for the binding of U2 snRNP to the pre-mRNA branch site. Purified U2AF comprises two polypeptides of 65 and 35 kd. We have performed biochemical complementation and immunological assays to characterize U2AF in greater detail. First, we use an extract lacking only U2AF activity to show that U2AF is an essential splicing factor. Second, we show that all U2AF activity in vitro resides in the 65 kd U2AF polypeptide. Third, based upon both immunological and functional criteria, we show that U2AF is evolutionarily conserved. Most significantly, a Drosophila melanogaster nuclear extract contains proteins that are antigenically related to both human U2AF polypeptides and can substitute for human U2AF in vitro. Finally, we show that U2AF has an unexpected intranuclear distribution. Although diffusely present throughout the nucleoplasm, U2AF is also concentrated in a small number (between one and five) of nuclear 'centers.' Th...

Pre-mRNA splicing, the removal of noncoding intron sequences from the pre-mRNA, is a
critical reaction in eukaryotic gene expression. Pre-mRNA splicing is carried out by a remarkable macromolecular machine, the spliceosome, which undergoes dynamic rearrangements of its RNA and protein components to assemble its catalytic center. While significant progress has been made in describing the ‘‘moving parts’’ of this machine, the mechanisms by which spliceosomal proteins mediate the ordered rearrangements within the spliceosome remain elusive. Here we explore recent evidence from proteomics studies revealing extensive post-translational modification
of splicing factors. While the functional significance of most of these modifications remains
to be characterized, we describe recent studies in which the roles of specific post-translational modifications of splicing factors have been characterized.

In this second review on chemokines, we focus on the polymorphisms and alternative splicings and on their consequences in disease. Because chemokines are key mediators in the pathogenesis of inflammatory, autoimmune, vascular and neoplastic disorders, a large number of studies attempting to relate particular polymorphisms of chemokines to given diseases have already been conducted, sometimes with contradictory results. Reviewing the published data, it becomes evident that some chemokine genes that are polymorphic have alleles that are found repeatedly, associated with disease of different aetiologies but sharing some aspects of pathogenesis. Among CXC chemokines, single nucleotide polymorphisms (SNPs) in the CXCL8 and CXCL12 genes stand out, as they have alleles associated with many diseases such as asthma and human immunodeficiency virus (HIV), respectively. Of CC chemokines, the stronger associations occur among alleles from SNPs in CCL2 and CCL5 genes and a number of inflammatory conditions. To understand how chemokines contribute to disease it is also necessary to take into account all the isoforms resulting from differential splicing. The first part of this review deals with polymorphisms and the second with the diversity of molecular species derived from each chemokine gene due to alternative splicing phenomena. The number of molecular species and the level of expression of each of them for every chemokine and for each functionally related group of chemokines reaches a complexity that requires new modelling algorithms akin to those proposed in systems biology approaches.

X-box binding protein 1 (XBP-1) is stimulated by endoplasmic reticulum stress as part of the unfolded protein response (UPR), which can promote apoptosis or cell survival. Non-conventional splicing, stimulated during the UPR, converts mRNA for “unspliced” XBP-1U to “spliced” XBP-1S mRNA. XBP-1 mRNA is oestrogen-responsive, but XBP-1S confers oestrogen independence and anti-oestrogen resistance to breast cancer cell lines. We therefore evaluated XBP-1 mRNA splicing as a factor in response of breast cancer patients to endocrine treatment. XBP-1 isoforms were measured by quantitative RT-PCR in 100 primary breast cancer patients treated with adjuvant tamoxifen (including 30 ERα-negative cases). In ERα-positive cases, levels of XBP-1U mRNA correlated with ERα mRNA levels and were lower in grade 3 tumors. Higher levels of XBP-1U mRNA were significantly associated with breast cancer survival (Log-rank p = 0.002; Cox hazard ratio (HR) 0.2, p = 0.005), independent of grade, size, nodal status and progesterone receptor status. However, in the full cohort, higher ratios of XBP-1S/XBP-1U mRNA (indicating enhanced splicing) were associated with poor survival (Log-rank p = 0.03; Cox HR 2.3, p = 0.03) and related factors: ERα-negative status, progesterone receptor negative status, grade 3 tumors and greater proliferation. Significant associations with poor outcome were also seen for XBP-1 splicing in ERα-positive cases. Our findings, that XBP-1 isoforms are differently associated with outcome of endocrine therapy for patients, can be explained by higher levels of dominant-negative XBP-1U favouring apoptosis of tumor cells and higher levels of XBP-1S increasing tumor survival. © 2008 Wiley-Liss, Inc.

The organization and biologically significant sequences of the entire mouse nerve growth factor (NGF) gene have been determined. The gene spans 45 kilobases and contains several small 5' exons. Transcription of the gene results in four different mRNA species, which can be accounted for by alternative splicing and independent initiation from two promoters. These transcripts encode proteins which have divergent N termini and the NGF moiety at their C termini. The levels of the various NGF transcripts have been determined in different tissues and throughout postnatal development. We have also examined the expression of these transcripts in the brain in response to specific early sensory deprivation. The results suggest that the expression of NGF mRNA during postnatal development is regulated independently of the formation of complex neural networks.

In recent years, oligonucleotide-based molecules have been intensely used to modulate gene expression. All these molecules share the common feature of being essentially impermeable over cellular membranes and they therefore require efficient delivery vectors. Cell-penetrating peptides are a group of delivery peptides that has been readily used for nucleic acid delivery. In particular, polyarginine and derivates thereof, i.e. the (RxR)4 peptide, have been applied with success both in vitro and in vivo. A major problem, however, with these arginine-rich peptides is that they frequently remain trapped in endosomal compartments following internalization. The activity of polyarginine has previously been improved by conjugation to a stearyl moiety. Therefore, we sought to investigate what impact such modification would have on the pre-clinically used (RxR)4 peptide for non-covalent delivery of plasmids and splice-correcting oligonucleotides (SCOs) and compare it with stearylated Arg9 and Lipofectamine™ 2000.We show that stearyl-(RxR)4 mediates efficient plasmid transfections in several cell lines and the expression levels are significantly higher than when using unmodified (RxR)4 or stearylated Arg9. Although the transfection efficiency is lower than with Lipofectamine™ 2000, we show that stearyl-(RxR)4 is substantially less toxic. Furthermore, using a functional splice-correction assay, we show that stearyl-(RxR)4 complexed with 2′-OMe SCOs promotes significant splice correction whereas stearyl-Arg9 fails to do so. Moreover, stearyl-(RxR)4 promotes dose-dependent splice correction in parity with (RxR)4-PMO covalent conjugates, but at least 10-times lower concentration. These features make this stearic acid modified analog of (RxR)4 an intriguing vector for future in vivo experiments.

The shuttling serine/arginine rich (SR) protein SRSF1 (previously known as SF2/ASF) is a splicing regulator that also activates translation in the cytoplasm. In order to dissect the gene network that is translationally regulated by SRSF1, we performed a high-throughput deep sequencing analysis of polysomal fractions in cells overexpressing SRSF1. We identified approximately 1500 mRNAs that are translational targets of SRSF1. These include mRNAs encoding proteins involved in cell cycle regulation, such as spindle, kinetochore, and M phase proteins, which are essential for accurate chromosome segregation. Indeed, we show that translational activity of SRSF1 is required for normal mitotic progression. Furthermore, we found that mRNAs that display alternative splicing changes upon SRSF1 overexpression are also its translational targets, strongly suggesting that SRSF1 couples pre-mRNA splicing and translation. These data provide insights on the complex role of SRSF1 in the control of gen...