650 research outputs found

    Purification and characterization of pre-mRNA splicing factor SF2 from HeLa cells

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    SF2, an activity necessary for 5' splice site cleavage and lariat formation during pre-mRNA splicing in vitro, has been purified to near homogeneity from HeLa cells. The purest fraction contains only two related polypeptides of 33 kD. This fraction is sufficient to complement an S100 fraction, which contains the remaining splicing factors, to splice several pre-mRNAs. The optimal amount of SF2 required for efficient splicing depends on the pre-mRNA substrate. SF2 is distinct from the hnRNP A1 and U1 snRNP a polypeptides, which are similar in size. Endogenous hnRNA copurifies with SF2, but this activity does not appear to have an essential RNA component. SF2 appear to be necessary for the assembly or stabilization of the earliest specific prespliceosome complex, although in the absence of other components, it can bind RNA in a nonspecific manner. SF2 copurifies with an activity that promotes the annealing of complementary RNAs. Thus, SF2 may promote specific RNA-RNA interactions between snRNAs and pre-mRNA, between complementary snRNA regions, and/or involving intramolecular pre-mRNA helices. Other purified proteins with RNA annealing activity cannot substitute for SF2 in the splicing reaction

    A specific subset of SR proteins shuttles continuously between the nucleus and the cytoplasm

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    The SR proteins constitute a large family of nuclear phosphoproteins required for constitutive pre-mRNA splicing. These factors also have global, concentration-dependent effects on alternative splicing regulation and this activity is antagonized by members of the hnRNP A/B family of proteins. We show here that whereas some human SR proteins are confined to the nucleus, three of them-SF2/ASF, SRp20, and 9G8-shuttle rapidly and continuously between the nucleus and the cytoplasm. By swapping the corresponding domains between shuttling and nonshuttling SR proteins, we show that the carboxy-terminal arginine/serine-rich (RS) domain is required for shuttling. This domain, however, is not sufficient to promote shuttling of an unrelated protein reporter, suggesting that stable RNA binding mediated by the RNA-recognition motifs may be required for shuttling. Consistent with such a requirement, a double point-mutation in RRM1 of SF2/ASF that impairs RNA binding prevents the protein from shuttling. In addition, we show that phosphorylation of the RS domain affects the shuttling properties of SR proteins. These findings show that different SR proteins have unique intracellular transport properties and suggest that the family members that shuttle may have roles not only in nuclear pre-mRNA splicing but also in mRNA transport, cytoplasmic events, and/or processes that involve communication between the nucleus and the cytoplasm

    Influence of Mo on the Fe:Mo:C nano-catalyst thermodynamics for single-walled carbon nanotube growth

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    We explore the role of Mo in Fe:Mo nanocatalyst thermodynamics for low-temperature chemical vapor deposition growth of single walled carbon nanotubes (SWCNTs). By using the size-pressure approximation and ab initio modeling, we prove that for both Fe-rich (~80% Fe or more) and Mo-rich (~50% Mo or more) Fe:Mo clusters, the presence of carbon in the cluster causes nucleation of Mo2C. This enhances the activity of the particle since it releases Fe, which is initially bound in a stable Fe:Mo phase, so that it can catalyze SWCNT growth. Furthermore, the presence of small concentrations of Mo reduce the lower size limit of low-temperature steady-state growth from ~0.58nm for pure Fe particles to ~0.52nm. Our ab initio-thermodynamic modeling explains experimental results and establishes a new direction to search for better catalysts.Comment: 7 pages, 3 figures. submitte

    Semiconductor saturable absorber mirror structures with low saturation fluence

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    We present two novel semiconductor saturable absorber mirror (SESAM) designs which can exhibit more than ten times lower saturation fluence than classical SESAM devices. Design considerations and characterization data are presented. These devices are particularly suited for passively mode-locked lasers with ultra-high repetition rate

    GHz QKD at telecom wavelengths using up-conversion detectors

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    We have developed a hybrid single photon detection scheme for telecom wavelengths based on nonlinear sum-frequency generation and silicon single-photon avalanche diodes (SPADs). The SPAD devices employed have been designed to have very narrow temporal response, i.e. low jitter, which we can exploit for increasing the allowable bit rate for quantum key distribution. The wavelength conversion is obtained using periodically poled Lithium niobate waveguides (W/Gs). The inherently high efficiency of these W/Gs allows us to use a continuous wave laser to seed the nonlinear conversion so as to have a continuous detection scheme. We also present a 1.27GHz qubit repetition rate, one-way phase encoding, quantum key distribution experiment operating at telecom wavelengths that takes advantage of this detection scheme. The proof of principle experiment shows a system capable of MHz raw count rates with a QBER less than 2% and estimated secure key rates greater than 100 kbit/s over 25 km.Comment: 12 pages, 7 figure

    175 GHz, 400-fs-pulse harmonically mode-locked surface emitting semiconductor laser

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    We report a harmonically mode-locked vertical external cavity surface emitting laser (VECSEL) producing 400 fs pulses at a repetition frequency of 175 GHz with an average output power of 300 mW. Harmonic mode-locking was established using a 300 µm thick intracavity single crystal diamond heat spreader in thermal contact with the front surface of the gain sample using liquid capillary bonding. The repetition frequency was set by the diamond microcavity and stable harmonic mode locking was achieved when the laser cavity length was tuned so that the laser operated on the 117th harmonic of the fundamental cavity. When an etalon placed intracavity next to the gain sample, but not in thermal contact was used pulse groups were observed. These contained 300 fs pulses with a spacing of 5.9 ps. We conclude that to achieve stable harmonic mode locking at repetition frequencies in the 100s of GHz range in a VECSEL there is a threshold pulse energy above which harmonic mode locking is achieved and below which groups of pulses are observed

    TSUNAMI: an antisense method to phenocopy splicing-associated diseases in animals

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    Antisense oligonucleotides (ASOs) are versatile molecules that can be designed to specifically alter splicing patterns of target pre-mRNAs. Here we exploit this feature to phenocopy a genetic disease. Spinal muscular atrophy (SMA) is a motor neuron disease caused by loss-of-function mutations in the SMN1 gene. The related SMN2 gene expresses suboptimal levels of functional SMN protein due to alternative splicing that skips exon 7; correcting this defect-e.g., with ASOs-is a promising therapeutic approach. We describe the use of ASOs that exacerbate SMN2 missplicing and phenocopy SMA in a dose-dependent manner when administered to transgenic Smn(-/-) mice. Intracerebroventricular ASO injection in neonatal mice recapitulates SMA-like progressive motor dysfunction, growth impairment, and shortened life span, with alpha-motor neuron loss and abnormal neuromuscular junctions. These SMA-like phenotypes are prevented by a therapeutic ASO that restores correct SMN2 splicing. We uncovered starvation-induced splicing changes, particularly in SMN2, which likely accelerate disease progression. These results constitute proof of principle that ASOs designed to cause sustained splicing defects can be used to induce pathogenesis and rapidly and accurately model splicing-associated diseases in animals. This approach allows the dissection of pathogenesis mechanisms, including spatial and temporal features of disease onset and progression, as well as testing of candidate therapeutics

    Isolated pseudo-RNA-recognition motifs of SR proteins can regulate splicing using a noncanonical mode of RNA recognition

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    Serine/arginine (SR) proteins, one of the major families of alternativesplicing regulators in Eukarya, have two types of RNA-recognition motifs (RRMs): a canonical RRM and a pseudo-RRM. Although pseudo-RRMs are crucial for activity of SR proteins, their mode of action was unknown. By solving the structure of the human SRSF1 pseudo-RRM bound to RNA, we discovered a very unusual and sequence-specific RNA-binding mode that is centered on one a-helix and does not involve the β-sheet surface, which typically mediates RNA binding by RRMs. Remarkably, this mode of binding is conserved in all pseudo-RRMs tested. Furthermore, the isolated pseudo- RRM is sufficient to regulate splicing of about half of the SRSF1 target genes tested, and the bound a-helix is a pivotal element for this function. Our results strongly suggest that SR proteins with a pseudo-RRM frequently regulate splicing by competing with, rather than recruiting, spliceosome components, using solely this unusual RRM

    Evidence that Myb-related CDC5 proteins are required for pre-mRNA splicing

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    The conserved CDC5 family of Myb-related proteins performs an essential function in cell cycle control at G(2)/M. Although c-Myb and many Myb-related proteins act as transcription factors, herein, we implicate CDC5 proteins in pre-mRNA splicing. Mammalian CDC5 colocalizes with pre-mRNA splicing factors in the nuclei of mammalian cells, associates with core components of the splicing machinery in nuclear extracts, and interacts with the spliceosome throughout the splicing reaction in vitro. Furthermore, genetic depletion of the homolog of CDC5 in Saccharomyces cerevisiae, CEF1. blocks the first step of pre-mRNA processing in vivo. These data provide evidence that eukaryotic cells require CDC5 proteins for pre-mRNA splicing

    RNA-sequencing of a mouse-model of spinal muscular atrophy reveals tissue-wide changes in splicing of U12-dependent introns

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    Spinal Muscular Atrophy (SMA) is a neuromuscular disorder caused by insufficient levels of the Survival of Motor Neuron (SMN) protein. SMN is expressed ubiquitously and functions in RNA processing pathways that include trafficking of mRNA and assembly of snRNP complexes. Importantly, SMA severity is correlated with decreased snRNP assembly activity. In particular, the minor spliceosomal snRNPs are affected, and some U12-dependent introns have been reported to be aberrantly spliced in patient cells and animal models. SMA is characterized by loss of motor neurons, but the underlying mechanism is largely unknown. It is likely that aberrant splicing of genes expressed in motor neurons is involved in SMA pathogenesis, but increasing evidence indicates that pathologies also exist in other tissues. We present here a comprehensive RNA-seq study that covers multiple tissues in an SMA mouse model. We show elevated U12-intron retention in all examined tissues from SMA mice, and that U12-dependent intron retention is induced upon siRNA knock-down of SMN in HeLa cells. Furthermore, we show that retention of U12-dependent introns is mitigated by ASO treatment of SMA mice and that many transcriptional changes are reversed. Finally, we report on missplicing of several Ca2+ channel genes that may explain disrupted Ca2+ homeostasis in SMA and activation of Cdk5
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