Biomedical subjects

A Mayeda

Publications and source records attributed to A Mayeda.

At least 37 records · Page 2Linked to original sources

PubMed1998

Effects of indirect light and propranolol on melatonin levels in normal human subjects.

An indirect lighting protocol was developed to measure nocturnal melatonin suppression by light in normal human subjects. Goals were to minimize both discomfort due to staring intensely at a bright light source, and behavioral variation due to wandering gaze. Subjects sat with a bank of five full-spectrum light sources placed behind them. Lights reflecting off the surfaces before each subject produced a hemisphere of light that measured 500 lx +/- 5%. Subjects retired to bed in darkness by midnight and then sat in the hemisphere of light from 02.00 h to 04.00 h. Blood for melatonin was drawn at 20-30-min intervals from midnight to 06.00 h. Plasma melatonin was measured by radioimmunoassay. The indirect lighting protocol was used to compare the effects of 500 lx light to dark (21 subjects) and to study varying light intensities from 300 to 2000 lx (7 subjects). We studied the effects of the sitting posture in very dim light of 20-30 lx (6 subjects). We also studied the effects of propranolol plus dark and propranolol plus 500 lx light on melatonin levels. Subjects received placebo, 10 mg propranolol or 40 mg propranolol orally at 23.00 h, and were then exposed to either the dark or light condition. Melatonin levels obtained with the indirect lighting protocol were consistent with studies using direct lighting; light of 500 lx significantly suppressed nocturnal melatonin and suppression was dose related between 300 and 2000 lx. Sitting in dim light had no significant effect on melatonin suppression when compared with the supine posture in the dark in six subjects. Propranolol caused a dose-dependent decrease in melatonin levels in both the dark and the light. There was no relationship between suppression of melatonin by propranolol and suppression by light.

Adolescent↗

PubMed1998

Distinct functions of the closely related tandem RNA-recognition motifs of hnRNP A1.

hnRNP A1 regulates alternative splicing by antagonizing SR proteins. It consists of two closely related, tandem RNA-recognition motifs (RRMs), followed by a glycine-rich domain. Analysis of variant proteins with duplications, deletions, or swaps of the RRMs showed that although both RRMs are required for alternative splicing function, each RRM plays distinct roles, and their relative position is important. Surprisingly, RRM2 but not RRM1 could support this function when duplicated, despite their very similar structure. Specific RNA binding and annealing are not sufficient for hnRNP A1 alternative splicing function. These observations, together with phylogenetic and structural data, suggest that the two RRMs are quasi-symmetric but functionally nonequivalent modules that evolved as components of a single bipartite domain.

Alternative Splicing↗

PubMed1998

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 SF2/ASF and used them to measure the abundance of SF2/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 SF2/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 SF2/ASF to that of its antagonist, hnRNP A1, 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.

Adenoviridae↗

PubMed1997

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 UPI 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 A 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 A, and a data set to 2.0 A has been collected. Diffraction data from one platinum and two mercury derivatives have also been collected.

Journal Article↗

PubMed1997

Initial genomic scan of the NIMH genetics initiative bipolar pedigrees: chromosomes 3, 5, 15, 16, 17, and 22.

As part of the four-center NIMH Genetics Initiative on Bipolar Disorder we carried out a genomic scan of chromosomes 3, 5, 15, 16,17, and 22. Genotyping was performed on a set of 540 DNAs from 97 families, enriched for affected relative pairs and parents where available. We report here the results of the initial 74 markers that have been typed on this set of DNAs. The average distance between markers (theta) was 12.3 cM. Nonparametric analysis of excess allele sharing among affected sibling pairs used the SIBPAL program of the S.A.G.E. package to test three hierarchical models of affected status. D16S2619 gave some evidence of linkage to bipolar disorder, with P = 0.006 for Model II (in which bipolar 1, bipolar 2 and schizoaffective-bipolar type individuals are considered affected). Nearby markers also showed increased allele sharing. A second interesting region was toward the telomere of chromosome 5q, where D5S1456 and nearby markers showed increased allele sharing; for D5S1456, P = 0.05, 0.015 and 0.008 as the models of affected status become more broad. MOD score analysis also supported the possible presence of a susceptibility locus in this region of chromosome 5. A pair of adjacent markers on chromosome 3, D3S2405 and D3S3038, showed a modest increased allele sharing in the broad model. Several isolated markers had excess allele sharing at the P < 0.05 level under a single model. D15S217 showed a MOD score of 2.37 (P < 0.025). Multipoint analysis flagged the region of chromosome 22 around D22S533 as the most interesting. Thus, several regions showed modest evidence for linkage to bipolar disorder in this initial genomic scan of these chromosomes, including broad regions near previous reports of possible linkage.

Alleles↗

PubMed1997

Crystal structure of human UP1, the domain of hnRNP A1 that contains two RNA-recognition motifs.

BACKGROUND: Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is one of the most abundant core proteins of hnRNP complexes in metazoan nuclei. It behaves as a global regulator of alternative pre-mRNA splicing by antagonizing the activities of several serine/arginine-rich splicing factors (SR proteins), resulting in the activation of distal alternative 5' splice sites and skipping of optional exons. Purified hnRNP A1 has nucleic acid annealing activity. The protein also shuttles continuously between the nucleus and the cytoplasm, a process mediated by signals within its C-terminal glycine-rich domain. The N-terminal region of human hnRNP A1, termed unwinding protein 1 (UP1), contains two RNA-recognition motifs (RRMs), RRM1 and RRM2. Understanding the structural elements by which hnRNP A1 interacts with RNA will have broad implications for studies of RNA processing. RESULTS: The crystal structure of UP1 has been determined to 1.9 A resolution. Each RRM independently adopts the characteristic RRM fold, consisting of a four-stranded antiparallel beta-pleated sheet and two alpha helices packed on one side of the beta sheet. The two RRMs are antiparallel and held in close contact, mainly by two Arg-Asp ion pairs. As a result, the two four-stranded beta sheets are brought together to form an extended RNA-binding surface. A segment of the linker connecting the two RRMs is flexible in the absence of bound RNA, but the general location of the linker suggests that it can make direct contacts with RNA. Comparison with other RRM structures indicates that a short 310 helix, found immediately N-terminal to the first beta strand in RRM1, may interact with RNA directly. CONCLUSIONS: The RRM is one of the most common and best characterized RNA-binding motifs. In certain cases, one RRM is sufficient for sequence-specific and high affinity RNA binding; but in other cases, synergy between several RRMs within a single protein is required. This study shows how two RRMs are organized in a single polypeptide. The two independently folded RRMs in UP1 are held together in a fixed geometry, enabling the two RRMs to function as a single entity in binding RNA, and so explaining the synergy between the RRMs. The UP1 structure also suggests that residues which lie outside of the RRMs can make potentially important interactions with RNA.

Amino Acid Sequence↗

PubMed1997

RNA splicing specificity determined by the coordinated action of RNA recognition motifs in SR proteins.

Pre-mRNA splicing requires a large number of RNA-binding proteins that have one or more RNA-recognition motifs (RRMs). Among these is the SR protein family, whose members are essential for splicing and are able to commit pre-mRNAs to the splicing pathway with overlapping but distinct substrate specificity. Some SR proteins, such as SC35, contain an N-terminal RRM and a C-terminal arginine/serine-rich (RS) domain, whereas others, such as SF2/ASF, also contain a second, atypical RRM. Although both the RRMs and the RS domain of SR proteins are required for constitutive splicing, it is unclear which domain(s) defines their substrate specificity, and whether two RRMs in a given SR protein function independently or act coordinately. Using domain swaps between SC35 and SF2/ASF and a functional commitment assay, we demonstrate that individual domains are functional modules, RS domains are interchangeable, and substrate specificity is defined by the RRMs. The atypical RRM of SF2/ASF does not appear to function alone in splicing, but can either activate or suppress the splicing specificity of an N-terminal RRM. Therefore, multiple RRMs in SR proteins act coordinately to achieve a unique spectrum of pre-mRNA substrate specificity.

Binding Sites↗

PubMed1997

RCC1 and nuclear organization.

We have examined the effect of RCC1 function on the nuclear organization of pre-mRNA splicing factors and poly(A)+ RNA in the tsBN2 cells, a RCC1 temperature-sensitive mutant cell line. We have found that at 4-6 h after shifting cells from the permissive temperature (32.5 degrees C) to the restrictive temperature (39.5 degrees C), both small nuclear ribonucleoprotein particles and a general splicing factor SC35 reorganized into 4-10 large round clusters in the nucleus, as compared with the typical speckled distribution seen in cells at the permissive temperature. In situ hybridization to poly(A)+ RNA resulted in a similar pattern. Examination by double labeling demonstrated that the redistribution of splicing factors coincides with that of poly(A)+ RNA. Such changes in the nuclear organization of splicing factors and poly(A)+ RNA were not the result of the temperature shift or of chromatin condensation. Cellular transcription was not significantly altered in these cells and extracts made from both the permissive and restrictive temperature were splicing competent. Electron microscopic examination demonstrated that the large clusters containing both splicing factors and poly(A)+ RNA were fused interchromatin granule clusters. In addition, small electron-dense dot-like structures measuring approximately 80 nm in diameter were also observed, most of which are accumulated in enlarged interchromatin granule clusters in the nucleoplasm of RCC1- cells. In spite of the significant changes observed in the nucleoplasm, relatively little alteration was observed in nucleolar structure by both light and electron microscopic examination. The above observations suggest that the RCC1 protein directly or indirectly regulates the organization of splicing components and poly(A)+ RNA in the cell nucleus and that RCC1 may play a role in nuclear organization.

Animals↗

PubMed1996

Pre-mRNA splicing in plants: characterization of Ser/Arg splicing factors.

The fact that animal introns are not spliced out in plants suggests that recognition of pre-mRNA splice sites differs between the two kingdoms. In plants, little is known about proteins required for splicing, as no plant in vitro splicing system is available. Several essential splicing factors from animals, such as SF2/ASF and SC-35, belong to a family of highly conserved proteins consisting of one or two RNA binding domain(s) (RRM) and a C-terminal Ser/Arg-rich (SR or RS) domain. These animal SR proteins are required for splice site recognition and spliceosome assembly. We have screened for similar proteins in plants by using monoclonal antibodies specific for a phosphoserine epitope of the SR proteins (mAb1O4) or for SF2/ASF. These experiments demonstrate that plants do possess SR proteins, including SF2/ASF-like proteins. Similar to the animal SR proteins, this group of proteins can be isolated by two salt precipitations. However, compared to the animal SR proteins, which are highly conserved in size and number, SR proteins from Arabidopsis, carrot, and tobacco exhibit a complex pattern of intra- and interspecific variants. These plant SR proteins are able to complement inactive HeLa cell cytoplasmic S1OO extracts that are deficient in SR proteins, yielding functional splicing extracts. In addition, plant SR proteins were active in a heterologous alternative splicing assay. Thus, these plant SR proteins are authentic plant splicing factors.

Animals↗

PubMed1995

Identification and characterization of three members of the human SR family of pre-mRNA splicing factors.

SR proteins have a characteristic C-terminal Ser/Arg-rich repeat (RS domain) of variable length and constitute a family of highly conserved nuclear phosphoproteins that can function as both essential and alternative pre-mRNA splicing factors. We have cloned a cDNA encoding a novel human SR protein designated SRp30c, which has an unusually short RS domain. We also cloned cDNAs encoding the human homologues of Drosophila SRp55/B52 and rat SRp40/HRS. Recombinant proteins expressed from these cDNAs are active in constitutive splicing, as shown by their ability to complement a HeLa cell S100 extract deficient in SR proteins. Additional cDNA clones reflect extensive alternative splicing of SRp40 and SRp55 pre-mRNAs. The predicted protein isoforms lack the C-terminal RS domain and might be involved in feedback regulatory loops. The ability of human SRp30c, SRp40 and SRp55 to modulate alternative splicing in vivo was compared with that of other SR proteins using a transient contransfection assay. The overexpression of individual SR proteins in HeLa cells affected the choice of alternative 5' splice sites of adenovirus E1A and/or human beta-thalassemia reporters. The resulting splicing patterns were characteristic for each SR protein. Consistent with the postulated importance of SR proteins in alternative splicing in vivo, we demonstrate complex changes in the levels of mRNAs encoding the above SR proteins upon T cell activation, concomitant with changes in the expression of alternatively spliced isoforms of CD44 and CD45.

Alternative Splicing↗

PubMed1994

Function of conserved domains of hnRNP A1 and other hnRNP A/B proteins.

hnRNP A1 is a pre-mRNA binding protein that antagonizes the alternative splicing activity of splicing factors SF2/ASF or SC35, causing activation of distal 5' splice sites. The structural requirements for hnRNP A1 function were determined by mutagenesis of recombinant human hnRNP A1. Two conserved Phe residues in the RNP-1 submotif of each of two RNA recognition motifs appear to be involved in specific RNA-protein interactions and are essential for modulating alternative splicing. These residues are not required for general pre-mRNA binding or RNA annealing activity. The C-terminal Gly-rich domain is necessary for alternative splicing activity, for stable RNA binding and for optimal RNA annealing activity. hnRNP A1B, which is an alternatively spliced isoform of hnRNP A1 with a longer Gly-rich domain, binds more strongly to pre-mRNA but has only limited alternative splicing activity. In contrast, hnRNP A2 and B1, which have 68% amino acid identity with hnRNP A1, bind more weakly to pre-mRNA and have stronger splice site switching activities than hnRNP A1. We propose that specific combinations of antagonistic hnRNP A/B and SR proteins are involved in regulating alternative splicing of distinct subsets of cellular premRNAs.

Amino Acid Sequence↗

PubMed1994

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.

Autoradiography↗

PubMed1994

The C-protein tetramer binds 230 to 240 nucleotides of pre-mRNA and nucleates the assembly of 40S heterogeneous nuclear ribonucleoprotein particles.

A series of in vitro protein-RNA binding studies using purified native (C1)3C2 and (A2)3B1 tetramers, total soluble heterogeneous nuclear ribonucleoprotein (hnRNP), and pre-mRNA molecules differing in length and sequence have revealed that a single C-protein tetramer has an RNA site size of 230 to 240 nucleotides (nt). Two tetramers bind twice this RNA length, and three tetramers fold monoparticle lengths of RNA (700 nt) into a unique 19S triangular complex. In the absence of this unique structure, the basic A- and B-group proteins bind RNA to form several different artifactual structures which are not present in preparations of native hnRNP and which do not function in hnRNP assembly. Three (A2)3B1 tetramers bind the 19S complex to form a 35S assembly intermediate. Following UV irradiation to immobilize the C proteins on the packaged RNA, the 19S triangular complex is recovered as a remnant structure from both native and reconstituted hnRNP particles. C protein-RNA complexes composed of three, six, or nine tetramers (one, two, or three triangular complexes) nucleate the stoichiometric assembly of monomer, dimer, and trimer hnRNP particles. The binding of C-protein tetramers to RNAs longer than 230 nt is through a self-cooperative combinatorial mode. RNA packaged in the 19S complex and in 40S hnRNP particles is efficiently spliced in vitro. These findings demonstrate that formation of the triangular C protein-RNA complex is an obligate first event in the in vitro and probably the in vivo assembly the 40S hnRNP core particle, and they provide insight into the mechanism through which the core proteins package 700-nt increments of RNA. These findings also demonstrate that unless excluded by other factors, the C proteins are likely to be located along the length of nascent transcripts.

Binding Sites↗

PubMed1993

Pathways for selection of 5' splice sites by U1 snRNPs and SF2/ASF.

We have used protection against ribonuclease H to investigate the mechanisms by which U1 small nuclear ribonucleoprotein particles (snRNPs) determine the use of two alternative 5' splice sites. The initial binding of U1 snRNPs to alternative consensus splice sites was indiscriminate, and on a high proportion of pre-mRNA molecules both sites were occupied simultaneously. When the sites were close, this inhibited splicing. We propose that double occupancy leads to the use of the downstream site for splicing and that this is the cause of the proximity effect seen with strong alternative splice sites. This model predicts that splicing to an upstream site of any strength requires a low affinity of U1 snRNPs for the downstream site. This prediction was tested both by cleaving the 5' end of U1 snRNA and by altering the sequence of the downstream site of an adenovirus E1A gene. The enhancement of downstream 5' splice site use by splicing factor SF2/ASF appears to be mediated by an increase in the strength of U1 snRNP binding to all sites indiscriminately.

Adenovirus E1A Proteins↗

PubMed1993

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.

Alternative Splicing↗

PubMed1993

Modulation of exon skipping and inclusion by heterogeneous nuclear ribonucleoprotein A1 and pre-mRNA splicing factor SF2/ASF.

The essential splicing factor SF2/ASF and the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) modulate alternative splicing in vitro of pre-mRNAs that contain 5' splice sites of comparable strengths competing for a common 3' splice site. Using natural and model pre-mRNAs, we have examined whether the ratio of SF2/ASF to hnRNP A1 also regulates other modes of alternative splicing in vitro. We found that an excess of SF2/ASF effectively prevents inappropriate exon skipping and also influences the selection of mutually exclusive tissue-specific exons in natural beta-tropomyosin pre-mRNA. In contrast, an excess of hnRNP A1 does not cause inappropriate exon skipping in natural constitutively or alternatively spliced pre-mRNAs. Although hnRNP A1 can promote alternative exon skipping, this effect is not universal and is dependent, e.g., on the size of the internal alternative exon and on the strength of the polypyrimidine tract in the preceding intron. With appropriate alternative exons, an excess of SF2/ASF promotes exon inclusion, whereas an excess of hnRNP A1 causes exon skipping. We propose that in some cases the ratio of SF2/ASF to hnRNP A1 may play a role in regulating alternative splicing by exon inclusion or skipping through the antagonistic effects of these proteins on alternative splice site selection.

Animals↗

PubMed1992

General splicing factors SF2 and SC35 have equivalent activities in vitro, and both affect alternative 5' and 3' splice site selection.

The human pre-mRNA splicing factors SF2 and SC35 have similar electrophoretic mobilities, and both of them contain an N-terminal ribonucleoprotein (RNP)-type RNA-recognition motif and a C-terminal arginine/serine-rich domain. However, the two proteins are encoded by different genes and display only 31% amino acid sequence identity. Here we report a systematic comparison of the splicing activities of recombinant SF2 and SC35. We find that either protein can reconstitute the splicing activity of S100 extracts and of SC35-immunodepleted nuclear extracts. Previous studies revealed that SF2 influences alternative 5' splice site selection in vitro, by favoring proximal over distal 5' splice sites, and that the A1 protein of heterogeneous nuclear RNP counteracts this effect. We now show that SC35 has a similar effect on competing 5' splice sites and is also antagonized by A1 protein. In addition, we report that both SF2 and SC35 also favor the proximal site in a pre-mRNA containing duplicated 3' splice sites, but this effect is not modulated by A1. We conclude that SF2 and SC35 are distinct splicing factors, but they display indistinguishable splicing activities in vitro.

Animals↗

PubMed1992

Two members of a conserved family of nuclear phosphoproteins are involved in pre-mRNA splicing.

Monoclonal antibody 104 recognizes a subset of amphibian nuclear granules (B-snurposomes) and active sites of RNA polymerase II transcription in vertebrates and invertebrates. Monoclonal antibody 104 reacts with a set of nuclear serine- and arginine-rich phosphoproteins (SR family) with strikingly conserved apparent molecular masses. The most abundant family members in human (SRp33) and Drosophila (SRp55) cell lines can replace one another as essential splicing factors in a human cell-free system. Each of these polypeptides can functionally replace human SF2, an essential splicing factor that also regulates 5' splice site selection of alternatively spliced pre-mRNAs in vitro. Drosophila SRp55 also functions as an alternative splicing factor in the human cell-free system. Analysis of cloned cDNAs shows that SRp55 and SF2 are highly related and reveals regions of similarity to genetically defined regulators of alternative splicing in Drosophila. These results suggest that the conserved SR family of phosphoproteins, which includes SRp55 and SF2, is involved in constitutive pre-mRNA splicing and in the specificity of alternative splice site selection.

Amino Acid Sequence↗