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

Regulated tissue-specific expression of antagonistic pre-mRNA splicing factors

The SR proteins are essential metazoan pre-mRNA splicing factors that can also influence the selection of alternative 5' splice sites in a concentration-dependent manner. Their activity in alternative splicing in vitro is antagonized by members of the hnRNP A/B family of proteins. The opposite effects of members of these two families of antagonistic splicing factors in vitro and upon overexpression in vivo suggest that changes in their relative levels may be a natural mechanism for the regulation of alternative splicing in vivo. One prediction of this model is that the ratios of these antagonists should vary in different cell types and in other situations in which cellular or viral transcripts are differentially spliced. We raised monoclonal antibodies specific for SFS/ASF and used them to measure the abundance of SFS/ASF protein and its isoforms, its phosphorylation state in vivo and during splicing in vitro, and its association with the spliceosome. SF2/ASF exists predominantly or exclusively in a highly phosphorylated state in vivo in all cell types examined, and unphosphorylated protein was not detectable. Unphosphorylated recombinant SFS/ASF becomes rapidly phosphorylated under splicing conditions in HeLa cell extracts and associates stably with one or more exons of beta-globin pre-mRNA. This interaction appears to persist through the splicing reaction and SF2/ASF remains bound to spliced mRNA. We compared the distribution of SFS/ASF to that of its antagonist, hnRNP Al, in different rat tissues and in immortal and transformed cell lines. We found that the protein levels of these antagonistic splicing factors vary naturally over a very wide range, supporting the notion that changes in the ratio of these proteins can affect alternative splicing of a variety of pre-mRNAs in vivo

A QTL on mouse chromosome 12 for the genetic variance in free-running circadian period between inbred strains of mice

<p>Abstract</p> <p>Background</p> <p>Many genes control circadian period in mice. Prior studies suggested a quantitative trait locus (QTL) on proximal mouse chromosome 12 for interstrain differences in circadian period. Since the B6.D2N<it>Ahr</it>^<it>d</it>/J strain has DBA/2 alleles for a portion of proximal chromosome 12 introgressed onto its C57BL/6J background, we hypothesized that these mice would have a shorter circadian period than C57BL/6J mice.</p> <p>Methods</p> <p>We compared circadian phenotypes of B6.D2N<it>Ahr</it>^<it>d</it>/J and C57BL/6 mice: period of general locomotor activity in constant dark and rest/activity pattern in alternating light and dark. We genotyped the B6.D2N<it>Ahr</it>^<it>d</it>/J mice to characterize the size of the genomic insert. To aid in identifying candidate quantitative trait genes we queried databases about the resident SNPs, whole brain gene expression in C57BL/6J versus DBA/2J mice, and circadian patterns of gene expression.</p> <p>Results</p> <p>The B6.D2N<it>Ahr</it>^<it>d</it>/J inbred mice have a shorter circadian period of locomotor activity than the C57BL/6J strain. Furthermore, the genomic insert is associated with another phenotype: the mean phase of activity minimum in the dark part of a light-dark lighting cycle. It was one hour later than in the background strain. The B6.D2N<it>Ahr</it>^<it>d</it>/J mice have a DBA/2J genomic insert spanning 35.4 to 41.0 megabase pairs on Chromosome 12. The insert contains 15 genes and 12 predicted genes. In this region <it>Ahr </it>(arylhydrocarbon receptor) and <it>Zfp277 </it>(zinc finger protein 277) both contain non-synonymous SNPs. <it>Zfp277 </it>also showed differential expression in whole brain and was cis-regulated. Three genes and one predicted gene showed a circadian pattern of expression in liver, including <it>Zfp277</it>.</p> <p>Conclusion</p> <p>We not only fine-mapped the QTL for circadian period on chromosome 12 but found a new QTL there as well: an association with the timing of the nocturnal activity-minimum. Candidate quantitative trait genes in this QTL are zinc finger protein 277 and arylhydrocarbon receptor. Arylhydrocarbon receptor is structurally related to <it>Bmal1</it>, a canonical clock gene.</p

A regional ground motion excitation/attenuation model for the San Francisco region

By using small-to-moderate-sized earthquakes located within ~200 km of San Francisco, we characterize the scaling of the ground motions for frequencies ranging between 0.25 and 20 Hz, obtaining results for geometric spreading, Q(f), and site parameters using the methods of Mayeda et al. (2005) and Malagnini et al. (2004). The results of the analysis show that, throughout the Bay Area, the average regional attenuation of the ground motion can be modeled with a bilinear geometric spreading function with a 30 km crossover distance, coupled to an anelastic function exp(-pi*f*r/V*Q(f)) , where: Q(f)=180f^0.42. A body-wave geometric spreading, g(r)= r^-1.0, is used at short hypocentral distances (r < 30 km), whereas g(r)= r^-0.6 fits the attenuation of the spectral amplitudes at hypocentral distances beyond the crossover. The frequency-dependent site effects at 12 of the Berkeley Digital Seismic Network (BDSN) stations were evaluated in an absolute sense using coda-derived source spectra. Our results show: i) the absolute site response for frequencies ranging between 0.3 Hz and 2.0 Hz correlate with independent estimates of the local magnitude residuals (dML) for each of the stations; ii) moment-magnitudes (MW) derived from our path and site-corrected spectra are in excellent agreement with those independently derived using full-waveform modeling as well as coda-derived source spectra; iii) we use our weak-motion-based relationships to predict motions region wide for the Loma Prieta earthquake, well above the maximum magnitude spanned by our data set, on a completely different set of stations. Results compare well with measurements taken at specific NEHRP site classes; iv) an empirical, magnitude-dependent scaling was necessary for the Brune stress parameter in order to match the large magnitude spectral accelerations and peak ground velocities with our weak-motion-based model

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

Disruption of pre-mRNA splicing in vivo results in reorganization of splicing factors

We have examined the functional significance of the organization of pre-mRNA splicing factors in a speckled distribution in the mammalian cell nucleus. Upon microinjection into living cells of oligonucleotides or antibodies that inhibit pre-mRNA splicing in vitro, we observed major changes in the organization of splicing factors in vivo. Interchromatin granule clusters became uniform in shape, decreased in number, and increased in both size and content of splicing factors, as measured by immunofluorescence. These changes were transient and the organization of splicing factors returned to their normal distribution by 24 h following microinjection. Microinjection of these oligonucleotides or antibodies also resulted in a reduction of transcription in vivo, but the oligonucleotides did not inhibit transcription in vitro. Control oligonucleotides did not disrupt splicing or transcription in vivo. We propose that the reorganization of splicing factors we observed is the result of the inhibition of splicing in vivo

A Regional Ground Motion Excitation attenuation Model for the San Francisco Region

By using small-to-moderate-sized earthquakes located within ~200 km of San Francisco, we characterize the scaling of the ground motions for frequencies ranging between 0.25 and 20 Hz, obtaining results for geometric spreading, Q(f), and site parameters using the methods of Mayeda et al. (2005) and Malagnini et al. (2004). The results of the analysis show that, throughout the Bay Area, the average regional attenuation of the ground motion can be modeled with a bilinear geometric spreading function with a 30 km crossover distance, coupled to an anelastic function ! exp " #fr $Q( f ) % & ' ( ) * , where: Q(f)=180 f 0.42. A body-wave geometric spreading, g(r)= r -1.0, is used at short hypocentral distances (r < 30 km), whereas g(r)= r -0.6 fits the attenuation of the spectral amplitudes at hypocentral distances beyond the crossover. The frequency-dependent site effects at 12 of the Berkeley Digital Seismic Network (BDSN) stations were evaluated in an absolute sense using coda-derived source spectra. Our results show: i) the absolute site response for frequencies ranging between 0.3 Hz and 2.0 Hz correlate with independent estimates of the local magnitude residuals (dML) for each of the stations; ii) moment-magnitudes (MW) derived from our path and sitecorrected spectra are in excellent agreement with those independently derived using fullwaveform modeling as well as coda-derived source spectra; iii) we use our weak-motionbased relationships to predict motions region wide for the Loma Prieta earthquake, well above the maximum magnitude spanned by our data set, on a completely different set of stations. Results compare well with measurements taken at specific NEHRP site classes; iv) an empirical, magnitude-dependent scaling was necessary for the Brune stress parameter in order to match the large magnitude spectral accelerations and peak ground velocities with our weak-motion-based model

Evidence for the function of an exonic splicing enhancer after the first catalytic step of pre-mRNA splicing

Exonic splicing enhancers (ESEs) activate pre-mRNA splicing by promoting the use of the flanking splice sites, They are recognized by members of the serine/arginine-rich (SR) family of proteins, such as splicing factor 2/alternative splicing factor (SF2/ASF), which recruit basal splicing factors to form the initial complexes during spliceosome assembly. The in vitro splicing kinetics of an ESE-dependent IgM pre-mRNA suggested that an SF2/ASF-specific ESE has additional functions later in the splicing reaction, after the completion of the first catalytic step. A bimolecular exon ligation assay, which physically uncouples the first and second catalytic steps of splicing in a trans-splicing reaction, was adapted to test the function of the ESE after the first step. A 3' exon containing the SF2/ASF-specific ESE underwent bimolecular exon ligation, whereas 3' exons without the ESE or with control sequences did not. The ESE-dependent trans-splicing reaction occurred after inactivation of U1 or U2 small nuclear ribonucleoprotein particles, compatible with a functional assay for events after the first step of splicing. The ESE-dependent step appears to take place before the ATP-independent part of the second catalytic step. Bimolecular exon ligation also occurred in an S100 cytosolic extract, requiring both the SF2/ASF-dependent ESE and complementation with SF2/ASF, These data suggest that some ESEs can act late in the splicing reaction, together with appropriate SR proteins, to enhance the second catalytic step of splicing