The RS4;11 cell line as a model for leukaemia with t(4;11)(q21;q23): Revised characterisation of cytogenetic features.

Background: Haematological malignancies harbouring rearrangements of the KMT2A gene represent a unique subtype of leukaemia, with biphenotypic clinical manifestations, a rapid and aggressive onset, and a generally poor prognosis. Chromosomal translocations involving KMT2A often cause the formation of oncogenic fusion genes, such as the most common translocation t(4;11)(q21;q23) producing the KMT2A-AFF1 chimera.

Aim: The aim of this study was to confirm and review the cytogenetic and molecular features of the KMT2A-rearranged RS4;11 cell line and put those in context with other reports of cell lines also harbouring a t(4;11) rearrangement.

The rare nonsense mutation in p53 triggers alternative splicing to produce a protein capable of inducing apoptosis.

P53 protein is more frequently mutated in human tumours compared with the other proteins. While the majority of the p53 mutations, especially within its DNA-binding domain, lead to the loss of the wild-type function, there are accumulating data demonstrating that the p53 mutants gain tumour promoting activities; the latter triggers a revitalised interest in functional analysis of the p53 mutants. A systematic screening for p53 mutations in surgical materials from patients with glioma revealed a 378C>G mutation that creates a stop codon at the position of amino acid residue 126.

The 35S U5 snRNP Is Generated from the Activated Spliceosome during In vitro Splicing.

Primary gene transcripts of eukaryotes contain introns, which are removed during processing by splicing machinery. Biochemical studies In vitro have identified a specific pathway in which introns are recognised and spliced out. This occurs by progressive formation of spliceosomal complexes designated as E, A, B, and C.

Exosomes are natural carriers of exogenous siRNA to human cells in vitro.

Background: Exosomes are nano-sized vesicles of endocytic origin that are involved in cell-to-cell communication including shuttle RNA, mainly mRNA and microRNA. As exosomes naturally carry RNA between cells, these particles might be useful in gene cancer therapy to deliver therapeutic short interfering RNA (siRNA) to the target cells. Despite the promise of RNA interference (RNAi) for use in therapy, several technical obstacles must be overcome.

Post-transcriptional spliceosomes are retained in nuclear speckles until splicing completion.

There is little quantitative information regarding how much splicing occurs co-transcriptionally in higher eukaryotes, and it remains unclear where precisely splicing occurs in the nucleus. Here we determine the global extent of co- and post-transcriptional splicing in mammalian cells, and their respective subnuclear locations, using antibodies that specifically recognize phosphorylated SF3b155 (P-SF3b155) found only in catalytically activated/active spliceosomes. Quantification of chromatin- and nucleoplasm-associated P-SF3b155 after fractionation of HeLa cell nuclei, reveals that ~80% of pre-mRNA splicing occurs co-transcriptionally.

Functional mammalian spliceosomal complex E contains SMN complex proteins in addition to U1 and U2 snRNPs.

Spliceosomes remove introns from primary gene transcripts. They assemble de novo on each intron through a series of steps that involve the incorporation of five snRNP particles and multiple non-snRNP proteins. In mammals, all the intermediate complexes have been characterized on one transcript (MINX), with the exception of the very first, complex E.

Progeria Research Day at Brunel University.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a severe premature aging syndrome that affects children. These children display characteristics associated with normal aging and die young usually from cardiovascular problems or stroke. Classical HGPS is caused by mutations in the gene encoding the nuclear structural protein lamin A.

A novel splice variant of the DNA-PKcs gene is associated with clinical and cellular radiosensitivity in a patient with xeroderma pigmentosum.

Background: Radiotherapy-induced DNA double-strand breaks (DSBs) are critical cytotoxic lesions. Inherited defects in DNA DSB repair pathways lead to hypersensitivity to ionising radiation, immunodeficiency and increased cancer incidence. A patient with xeroderma pigmentosum complementation group C, with a scalp angiosarcoma, exhibited dramatic clinical radiosensitivity following radiotherapy, resulting in death.

Isoforms of U1-70k control subunit dynamics in the human spliceosomal U1 snRNP.

Most human protein-encoding genes contain multiple exons that are spliced together, frequently in alternative arrangements, by the spliceosome. It is established that U1 snRNP is an essential component of the spliceosome, in human consisting of RNA and ten proteins, several of which are post-translationally modified and exist as multiple isoforms. Unresolved and challenging to investigate are the effects of these post translational modifications on the dynamics, interactions and stability of the particle.

Organization of core spliceosomal components U5 snRNA loop I and U4/U6 Di-snRNP within U4/U6.U5 Tri-snRNP as revealed by electron cryomicroscopy.

In eukaryotes, pre-mRNA exons are interrupted by large noncoding introns. Alternative selection of exons and nucleotide-exact removal of introns are performed by the spliceosome, a highly dynamic macromolecular machine. U4/U6.

A subset of human 35S U5 proteins, including Prp19, function prior to catalytic step 1 of splicing.

During catalytic activation of the spliceosome, snRNP remodeling events occur, leading to the formation of a 35S U5 snRNP that contains a large group of proteins, including Prp19 and CDC5, not found in 20S U5 snRNPs. To investigate the function of 35S U5 proteins, we immunoaffinity purified human spliceosomes that had not yet undergone catalytic activation (designated BDeltaU1), which contained U2, U4, U5, and U6, but lacked U1 snRNA. Comparison of the protein compositions of BDeltaU1 and activated B* spliceosomes revealed that, whereas U4/U6 snRNP proteins are stably associated with BDeltaU1 spliceosomes, 35S U5-associated proteins (which are present in B*) are largely absent, suggesting that they are dispensable for complex B formation.

Three-dimensional structure of a pre-catalytic human spliceosomal complex B.

Major structural changes occur in the spliceosome during its transition from the fully assembled complex B to the catalytically activated spliceosome. To understand the rearrangement, it is necessary to know the detailed three-dimensional structures of these complexes. Here, we have immunoaffinity-purified human spliceosomes (designated B Delta U1) at a stage after U4/U6.

Small nuclear ribonucleoprotein remodeling during catalytic activation of the spliceosome.

Major structural changes occur in the spliceosome during its activation just before catalyzing the splicing of pre-messenger RNAs (pre-mRNAs). Whereas changes in small nuclear RNA (snRNA) conformation are well documented, little is known about remodeling of small nuclear ribonucleoprotein (snRNP) structures during spliceosome activation. Here, human 45S activated spliceosomes and a previously unknown 35S U5 snRNP were isolated by immunoaffinity selection and were characterized by mass spectrometry.

Mammalian PRP4 kinase copurifies and interacts with components of both the U5 snRNP and the N-CoR deacetylase complexes.

A growing body of evidence supports the coordination of pre-mRNA processing and transcriptional regulation. We demonstrate here that mammalian PRP4 kinase (PRP4K) is associated with complexes involved in both of these processes. PRP4K is implicated in pre-mRNA splicing as the homologue of the Schizosaccharomyces pombe pre-mRNA splicing kinase Prp4p, and it is enriched in SC35-containing nuclear splicing speckles.

Human U4/U6.U5 and U4atac/U6atac.U5 tri-snRNPs exhibit similar protein compositions.

In the U12-dependent spliceosome, the U4atac/U6atac snRNP represents the functional analogue of the major U4/U6 snRNP. Little information is available presently regarding the protein composition of the former snRNP and its association with other snRNPs. In this report we show that human U4atac/U6atac di-snRNPs associate with U5 snRNPs to form a 25S U4atac/U6atac.

Protein 61K, encoded by a gene (PRPF31) linked to autosomal dominant retinitis pigmentosa, is required for U4/U6*U5 tri-snRNP formation and pre-mRNA splicing.

In each round of nuclear pre-mRNA splicing, the U4/U6*U5 tri-snRNP must be assembled from U4/U6 and U5 snRNPs, a reaction that is at present poorly understood. We have characterized a 61 kDa protein (61K) found in human U4/U6*U5 tri-snRNPs, which is homologous to yeast Prp31p, and show that it is required for this step. Immunodepletion of protein 61K from HeLa nuclear extracts inhibits tri-snRNP formation and subsequent spliceosome assembly and pre-mRNA splicing.