Splicing

"As Georges Méliès discovered to his astonished glee in the late nineteenth century, moving pictures do more than just record reality—they create one. Through stop-trick substitutions, multiple exposures, and splices, celluloid becomes a portal translucent not only to the projector’s bulb, but to the projections of the fantastic. For Argentine writer Adolfo Bioy Casares, such cinematic sleights-of-hand become so compelling that they promise to substitute a special effect for all of reality itself. In his 1940 novel The Invention of Morel, Bioy Casares represents two dissociated worlds that, although temporally estranged, are spliced together spatially through layered technological projections—creating the illusion of two lovers immortalized in a virtual union, played back without end. This Borgesian splicing of virtual and actual demonstrates the fantastical possibilities—and tremendous manipulative power—of the splice and the image overlay. In so doing, Bioy Casares raises striking questions about our ability to embrace fantastical projections and attempt to achieve satisfaction from virtual liaisons."

Three linen textiles discovered in the Ramon I rock shelter, in the Makhtesh Ramon, date from c. 3000 BCE, the Early Bronze Age II, based on direct dating of the textiles and their excavation contexts. In addition to adding important
and rare technological data on early textile manufacture, the contexts, in a rock shelter used for animal stabling, indicate long-distance trade in linen, penetrating
into the pastoral societies of the Negev.

Background

Most eukaryotic genes comprise exons and introns thus requiring the precise removal of introns from pre-mRNAs to enable protein biosynthesis. U2 and U12 spliceosomes catalyze this step by recognizing motifs on the transcript in order to remove the introns. A process which is dependent on precise definition of exon-intron borders by splice sites, which are consequently highly conserved across species. Only very few combinations of terminal dinucleotides are frequently observed at intron ends, dominated by the canonical GT-AG splice sites on the DNA level.
Results

Here we investigate the occurrence of diverse combinations of dinucleotides at predicted splice sites. Analyzing 121 plant genome sequences based on their annotation revealed strong splice site conservation across species, annotation errors, and true biological divergence from canonical splice sites. The frequency of non-canonical splice sites clearly correlates with their divergence from canonical ones indicating either an accumulation of probably neutral mutations, or evolution towards canonical splice sites. Strong conservation across multiple species and non-random accumulation of substitutions in splice sites indicate a functional relevance of non-canonical splice sites. The average composition of splice sites across all investigated species is 98.7% for GT-AG, 1.2% for GC-AG, 0.06% for AT-AC, and 0.09% for minor non-canonical splice sites. RNA-Seq data sets of 35 species were incorporated to validate non-canonical splice site predictions through gaps in sequencing reads alignments and to demonstrate the expression of affected genes.
Conclusion

We conclude that bona fide non-canonical splice sites are present and appear to be functionally relevant in most plant genomes, although at low abundance.

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.

Somatic mutations in spliceosome proteins lead to dysregulated RNA splicing and are observed in a variety of cancers. These genetic aberrations may offer a potential intervention point for targeted therapeutics. SF3B1, part of the U2 small nuclear RNP (snRNP), is targeted by splicing modulators, including E7107, the first to enter clinical trials, and, more recently, H3B-8800. Modulating splicing represents a first-in-class opportunity in drug discovery, and elucidating the structural basis for the mode of action opens up new possibilities for structure-based drug design. Here, we present the cryogenic electron microscopy (cryo-EM) structure of the SF3b subcomplex (SF3B1, SF3B3, PHF5A, and SF3B5) bound to E7107 at 3.95 Å. This structure shows that E7107 binds in the branch point adenosine-binding pocket, forming close contacts with key residues that confer resistance upon mutation: SF3B1 R1074H and PHF5A Y36C. The structure suggests a model in which splicing modulators interfere with branch point adenosine recognition and supports a substrate competitive mechanism of action (MOA). Using several related chemical probes, we validate the pose of the compound and support their substrate competitive MOA by comparing their activity against both strong and weak pre-mRNA substrates. Finally, we present functional data and structure–activity relationship (SAR) on the PHF5A R38C mutation that sensitizes cells to some chemical probes but not others. Developing small molecule splicing modulators represents a promising therapeutic approach for a variety of diseases, and this work provides a significant step in enabling structure-based drug design for these elaborate natural products. Importantly, this work also demonstrates that the utilization of cryo-EM in drug discovery is coming of age.

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.

An emerging trend in DNA computing consists of the algorithmic analysis of new molecular biology technologies, and in general of more effective tools to tackle computational biology problems. An algorithmic understanding of the interaction between DNA molecules becomes the focus of some research which was initially addressed to solve mathematical problems by processing data within biomolecules. In this paper a

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.

Mutations at nucleotides 654, 705, or 745 in intron 2 of the human -globin gene activate aberrant 3 and 5 splice sites within the intron and prevent correct splic- ing of -globin pre-mRNA, resulting in inhibition of -globin synthesis and in consequence -thalassemia. Transfec- tion of HeLa cells expressing the 3 thalas- semic mutants with modified U7 snRNA (U7.623), containing