Evolutionary impact of limited splicing fidelity in mammalian genes

The functional significance of most alternative splicing (AS) events, especially frame-shifting ones, has been controversial. Using human-mouse comparison, we demonstrate that frame-preserving AS events adapt and get fixed more rapidly than frame-shifting AS events; selection for smaller exon size is stronger in frame-preserving exons than in frame-shifting ones. These results suggest AS events introducing mild changes are generally favored during evolution and explain the excess of shorter, frame-preserving cassette exons in present mammalian genomes

The type 2C Ser/Thr phosphatase PP2C gamma is a pre-mRNA splicing factor

To identify activities involved in human pre-mRNA splicing, we have developed a procedure to separate HeLa cell nuclear extract into five complementing fractions. An activity called SCF1 was purified from one of these fractions by assaying for reconstitution of splicing in the presence of the remaining four fractions. A component of SCF1 is shown to be PP2C gamma, a type 2C Ser/Thr phosphatase of previously unknown function. Previous work suggested that dephosphorylation of splicing factors may be important for catalysis after spliceosome assembly, although the identities of the specific phosphatases involved remain unclear. Here we show that human PP2C gamma is physically associated with the spliceosome in vitro throughout the splicing reaction, but is first required during the early stages of spliceosome assembly for efficient formation of the A complex. The phosphatase activity is required for the splicing function of PP2C gamma, as an active site mutant does not support spliceosome assembly. The requirement for PP2C gamma is highly specific, as the closely related phosphatase PP2C alpha cannot substitute for PP2C gamma. Consistent with a role in splicing, PP2C gamma localizes to the nucleus in vivo. We conclude that at least one specific dephosphorylation event catalyzed by PP2C gamma is required for formation of the spliceosome

Crystallization and preliminary X-ray diffraction studies of UP1, the two-RRM domain of hnRNP A1

The N-terminal domain of hnRNP A1 protein, termed UP1, comprises two tandem RNA-recognition motifs, both of which are necessary for efficient RNA binding and for the alternative splicing activity of hnRNP A1. Recombinant human UP1 expressed in E. coli has been crystallized in space group P2(1) with unit-cell dimensions a = 37.94, b = 43.98, c = 55.64 Angstrom and beta = 93.9 degrees. The unit-cell volume is consistent with one UP1 molecule per asymmetric unit and a calculated 49% solvent content. The crystal diffraction limit is higher than 1.3 Angstrom, and a data set to 2.0 Angstrom has been collected. Diffraction data from one platinum and two mercury derivatives have also been collected

Control of Pre-mRNA Splicing by the General Splicing Factors PUF60 and U2AF65

Pre-mRNA splicing is a crucial step in gene expression, and accurate recognition of splice sites is an essential part of this process. Splice sites with weak matches to the consensus sequences are common, though it is not clear how such sites are efficiently utilized. Using an in vitro splicing-complementation approach, we identified PUF60 as a factor that promotes splicing of an intron with a weak 3' splice-site. PUF60 has homology to U2AF(65), a general splicing factor that facilitates 3' splice-site recognition at the early stages of spliceosome assembly. We demonstrate that PUF60 can functionally substitute for U2AF(65)in vitro, but splicing is strongly stimulated by the presence of both proteins. Reduction of either PUF60 or U2AF(65) in cells alters the splicing pattern of endogenous transcripts, consistent with the idea that regulation of PUF60 and U2AF(65) levels can dictate alternative splicing patterns. Our results indicate that recognition of 3' splice sites involves different U2AF-like molecules, and that modulation of these general splicing factors can have profound effects on splicing

Antisense-based therapy for the treatment of spinal muscular atrophy

One of the greatest thrills a biomedical researcher may experience is seeing the product of many years of dedicated effort finally make its way to the patient. As a team, we have worked for the past eight years to discover a drug that could treat a devastating childhood neuromuscular disease, spinal muscular atrophy (SMA). Here, we describe the journey that has led to a promising drug based on the biology underlying the disease

General splicing factor SF2/ASF promotes alternative splicing by binding to an exonic splicing enhancer

The general splicing factor SF2/ASF binds in a sequence-specific manner to a purine-rich exonic splicing enhancer (ESE) in the last exon of bovine growth hormone (bGH) pre-mRNA. More importantly, SF2/ASF stimulates in vitro splicing of bGH intron D through specific interaction with the ESE sequences. However, another general splicing factor, SC35, does not bind the ESE sequences and has no effect on bGH intron D splicing. Thus, one possible function of SF2/ASF in alternative and, perhaps, constitutive pre-mRNA splicing is to recognize ESE sequences. The stimulation of bGH intron D splicing by SF2/ASF is counteracted by the addition of hnRNP A1. The relative levels of SF2/ASF and hnRNP A1 influence the efficiency of bGH intron D splicing in vitro and may be the underlying mechanism of this alternative pre-mRNA processing event in vivo

RNA splicing at human immunodeficiency virus type 1 3 ' splice site A2 is regulated by binding of hnRNP A/B proteins to an exonic splicing silencer element

The synthesis of human immunodeficiency virus type 1 (HIV-1) mRNAs is a complex process by which more than 30 different mRNA species are produced by alternative splicing of a single primary RNA transcript. HIV-1 splice sites are used with significantly different efficiencies, resulting in different levels of mRNA species in infected cells. Splicing of Tat mRNA, which is present at relatively low levels in infected cells, is repressed by the presence of exonic splicing silencers (ESS) within the two tat coding exons (ESS2 and ESS3). These ESS elements contain the consensus sequence PyUAG. Here we show that the efficiency of splicing at 3 ' splice site A2, which is used to generate Vpr mRNA, is also regulated by the presence of an ESS (ESSV), which has sequence homology to ESS2 and ESS3. Mutagenesis of the three PyUAG motifs within ESSV increases splicing at splice site A2, resulting in increased Vpr mRNA levels and reduced skipping of the noncoding exon flanked by A2 and D3. The increase in Vpr mRNA levels and the reduced skipping also occur when splice site D3 is mutated toward the consensus sequence. By in vitro splicing assays, we show that ESSV represses splicing when placed downstream of a heterologous splice site. A1, A1(B), A2, and B1 hnRNPs preferentially bind to ESSV RNA compared to ESSV mutant RNA. Each of these proteins, when added back to HeLa cell nuclear extracts depleted of ESSV-binding factors, is able to restore splicing repression. The results suggest that coordinate repression of HIV-1 RNA splicing is mediated by members of the hnRNP A/B protein family

OLego: fast and sensitive mapping of spliced mRNA-Seq reads using small seeds

A crucial step in analyzing mRNA-Seq data is to accurately and efficiently map hundreds of millions of reads to the reference genome and exon junctions. Here we present OLego, an algorithm specifically designed for de novo mapping of spliced mRNA-Seq reads. OLego adopts a multiple-seed-and-extend scheme, and does not rely on a separate external aligner. It achieves high sensitivity of junction detection by strategic searches with small seeds ( approximately 14 nt for mammalian genomes). To improve accuracy and resolve ambiguous mapping at junctions, OLego uses a built-in statistical model to score exon junctions by splice-site strength and intron size. Burrows-Wheeler transform is used in multiple steps of the algorithm to efficiently map seeds, locate junctions and identify small exons. OLego is implemented in C++ with fully multithreaded execution, and allows fast processing of large-scale data. We systematically evaluated the performance of OLego in comparison with published tools using both simulated and real data. OLego demonstrated better sensitivity, higher or comparable accuracy and substantially improved speed. OLego also identified hundreds of novel micro-exons (<30 nt) in the mouse transcriptome, many of which are phylogenetically conserved and can be validated experimentally in vivo. OLego is freely available at http://zhanglab.c2b2.columbia.edu/index.php/OLego

Crystal structure of the two-RRM domain of hnRNP A1 (UP1) complexed with single-stranded telomeric DNA

Human hnRNP A1 is a versatile single-stranded nucleic acid-binding protein that functions in various aspects of mRNA maturation and in telomere length regulation. The crystal structure of UP1, the amino-terminal domain of human hnRNP A1 containing two RNA-recognition motifs (RRMs), bound to a 12-nucleotide single-stranded telomeric DNA has been determined at 2.1 Angstrom resolution. The structure of the complex reveals the basis for sequence-specific recognition of the single-stranded overhangs of human telomeres by hnRNP A1. It also provides insights into the basis for high-affinity binding of hnRNP A1 to certain RNA sequences, and for nucleic acid binding and functional synergy between the RRMs. In the crystal structure, a UP1 dimer binds to two strands of DNA, and each strand contacts RRM1 of one monomer and RRM2 of the other. The two DNA strands are antiparallel, and regions of the protein flanking each RRM make important contacts with DNA. The extensive protein-protein interface seen in the crystal structure of the protein-DNA complex and the evolutionary conservation of the interface residues suggest the importance of specific protein-protein interactions for the sequence-specific recognition of single-stranded nucleic acids. Models for regular packaging of telomere 3' overhangs and for juxtaposition of alternative 5' splice sites are proposed

Exergy analysis:Tthe effect of relative humidity, air temperature and effective clothing insulation on thermal comfort

abstract onl