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R H Singer

Publications and source records attributed to R H Singer.

At least 55 records · Page 3Linked to original sources
PubMed1997

Quantitative digital analysis of diffuse and concentrated nuclear distributions of nascent transcripts, SC35 and poly(A).

Digital imaging microscopy was used to analyze the spatial distribution and levels of newly synthesized RNA in relation to steady-state poly(A) RNA and to the splicing factor SC35. Transcription was monitored over time after microinjection of BrUTP and was detected using antibodies. Poly(A) RNA was detected with probes directly conjugated to fluorochromes, allowing direct detection of the hybrids. Objective methods were used to determine genuine signal. A defined threshold level to separate signal from noise was established for each nucleus. The nucleolus was used to determine poly(A) and SC35 background and the juxtanuclear cytoplasm was used for the BrUTP background. The remaining signal was segmented into high (concentrated) and low (diffuse) levels. Surprisingly, for all probes examined, most of the signal was not in concentrated areas, but rather was diffusely spread throughout the nucleoplasm. A minority (20-30%) of the SC35 signal was in concentrated areas ("speckles") and the rest was dispersed throughout the nucleoplasm. In addition, the concentrated areas had a mean intensity only twice the average. The amount and significance of the colocalization of the diffuse, or concentrated, areas of SC35 [or poly(A)] with BrUTP incorporation were analyzed. The image from one probe was translated with respect to the other in three dimensions to compare colocalization with random alignments. Both poly(A) and SC35 were found to have low colocalization with the total BrU signal. Sites of transcription were determined using an algorithm to find maxima of BrUTP signal within clusters. From 849 to as many as 3888 sites per nucleus were detected. A rim of hybridization to poly(A) coinciding with the nuclear envelope was eliminated by actinomycin treatment, suggesting that these transcripts were exiting from the nucleus. These results emphasize the importance of utilizing the full dynamic range of the image before drawing conclusions as to the distribution of nuclear components.

Algorithms↗
PubMed1997

mRNA and cytoskeletal filaments.

The localization of some mRNAs to distinct intracellular regions is achieved through interactions of the mRNA with cytoskeletal filaments. RNA-cytoskeletal interactions exist that influence the transport, anchoring and translation of mRNA. Recent analysis of RNA movements in living cells suggests the formation of RNA granules and their active transport along microtubules. The anchoring and translation of mRNA may be mediated by interactions with orthogonal networks of F-actin and elongation factor 1alpha.

Actin Cytoskeleton↗
PubMed1997

Characterization of a beta-actin mRNA zipcode-binding protein.

Localization of beta-actin mRNA to the leading edge of fibroblasts requires the presence of conserved elements in the 3' untranslated region of the mRNA, including a 54-nucleotide element which has been termed the "zipcode" (E. Kislauskis, X. Zhu, and R. H. Singer, J. Cell Biol. 127:441-451, 1994). In order to identify proteins which bind to the zipcode and possibly play a role in localization, we performed band-shift mobility assays, UV cross-linking, and affinity purification experiments. A protein of 68 kDa was identified which binds to the proximal (to the coding region) half of the zipcode with high specificity (ZBP-1). Microsequencing provided unique peptide sequences of approximately 15 residues each. Degenerate primers corresponding to the codons derived from the peptides were synthesized and used for PCR amplification. Screening of a chicken cDNA library resulted in isolation of several clones providing a DNA sequence encoding a 67.7-kDa protein with regions homologous to several RNA-binding proteins, such as hnRNP E1 and E2, and with consensus mRNA recognition motif with RNP1 and 2 motifs and a putative REV-like nuclear export signal. Antipeptide antibodies were raised in rabbits which bound to ZBP-1 and coimmunoprecipitated proteins of 120 and 25 kDa. The 120-kDa protein was also obtained by affinity purification with the RNA zipcode sequence, along with a 53-kDa protein, but the 25-kDa protein appeared only in immunoprecipitations. Mutation of one of the conserved sequences within the zipcode, an ACACCC element in its proximal half, greatly reduced its protein binding and localization properties. These data suggest that the 68-kDa ZBP-1 we have isolated and cloned is an RNA-binding protein that functions within a complex to localize beta-actin mRNA.

Actins↗
PubMed1997

Nuclear domains of the RNA subunit of RNase P.

The ribonucleoprotein enzyme RNase P catalyzes the 5' processing of pre-transfer RNA, and has also recently been implicated in pre-ribosomal RNA processing. In the present investigation, in situ hybridization revealed that RNase P RNA is present throughout the nucleus of mammalian cells. However, rhodamine-labeled human RNase P RNA microinjected into the nucleus of rat kidney (NRK) epithelial cells or human (HeLa) cells initially localized in nucleoli, and subsequently became more evenly distributed throughout the nucleus, similar to the steadystate distribution of endogenous RNase P RNA. Parallel microinjection and immunocytochemical experiments revealed that initially nucleus-microinjected RNase P RNA localized specifically in the dense fibrillar component of the nucleolus, the site of pre-rRNA processing. A mutant RNase P RNA lacking the To antigen binding domain (nucleotides 25-75) did not localize in nucleoli after nuclear microinjection. In contrast, a truncated RNase P RNA containing the To binding domain but lacking nucleotides 89-341 became rapidly localized in nucleoli following nuclear microinjection. However, unlike the full-length RNase P RNA, this 3' truncated RNA remained stably associated with the nucleoli and did not translocate to the nucleoplasm. These results suggest a nucleolar phase in the maturation, ribonucleoprotein assembly or function of RNase P RNA, mediated at least in part by the nucleolar To antigen. These and other recent findings raise the intriguing possibility of a bifunctional role of RNase P in the nucleus: catalyzing pre-ribosomal RNA processing in the nucleolus and pre-transfer RNA processing in the nucleoplasm.

Animals↗
PubMed1996

Triplet repeats and human disease.

In recent years a new mechanism of genetic disease has been discovered in which triplet DNA sequences (trinucleotides) expand either to interrupt or to compromise a gene. So far, ten genetic loci have been identified in which this event takes place. The presence of these expansions, sometimes containing thousands of repeated trinucleotides, provides a clue as to how processing of DNA and RNA may sometimes go awry. How this expansion in a single allele may exert a dominant effect and how the extent of the expansion increases the severity of the disease remain a mystery.

Animals↗
PubMed1995

Characterization of hybridization between synthetic oligodeoxynucleotides and RNA in living cells.

Cells internalized synthetic oligonucleotides (oligos) in culture. The hybridization of these molecules to target RNA in the living cell was subsequently detected and characterized after fixation of the cells, with or without previous detergent extraction. Hybridized oligo was distinguished from free oligo in the cell using an in situ reverse transcription technique. This assay exploited the ability of the hybridized oligo to prime synthesis of a specific cDNA strand; unhybridized oligo present in the cell could not act as a primer for reverse transcription. Phosphorothioate and fluorochrome-labeled phosphodiester oligo dT were found to enter cells rapidly and hybridize to poly (A) RNA within 30 min. Hybrids containing phosphorothioate oligo dT were detectable in cells after up to 4 h of efflux time. Phosphodiester bonded oligo dT containing covalently-linked fluorochromes appeared more stable in the cell than unmodified phosphodiester oligo dT; hybrids containing these oligos could be detected in cells as long as 18h after efflux began. The in situ transcription assay was also sensitive enough to detect hybridization of anti-actin oligos to actin mRNA in the cell. It is probable, therefore, that this assay can be used to help assess the efficacy of antisense oligos by their hybridization to a target mRNA in cells or tissues; hybridized oligos are more likely to induce a specific antisense effect. Additionally, this assay will help to identify probes that would be useful as stable hybridization tags to follow RNA movement in living cells.

Actins↗
PubMed1995

Foci of trinucleotide repeat transcripts in nuclei of myotonic dystrophy cells and tissues.

We have analyzed the intracellular localization of transcripts from the myotonin protein kinase (Mt-PK) gene in fibroblasts and muscle biopsies from myotonic dystrophy patients and normal controls. In affected individuals, a trinucleotide expansion in the gene results in the phenotype, the severity of which is proportional to the repeat length. A fluorochrome-conjugated probe (10 repeats of CAG) hybridized specifically to this expanded repeat. Mt-PK transcripts containing CTG repeat expansions were detected in the nucleus as bright foci in DM patient fibroblasts and muscle biopsies, but not from normal individuals. These foci represented transcripts from the Mt-PK gene since they simultaneously hybridized to fluorochrome-conjugated probes to the 5'-end of the Mt-PK mRNA. A single oligonucleotide probe to the repeat and the sense strand each conjugated to different fluorochromes revealed the gene and the transcripts simultaneously, and indicated that these focal concentrations (up to 13 per nucleus) represented predominately posttranscriptional RNA since only a single focus contained both the DNA and the RNA. This concentration of nuclear transcripts was diagnostic of the affected state, and may represent aberrant processing of the RNA.

Adult↗
PubMed1995

Spatial consequences of defective processing of specific yeast mRNAs revealed by fluorescent in situ hybridization.

This work introduces the first use of fluorescent in situ hybridization (FISH) to detect the distribution of specific transcripts in Saccharomyces cerevisiae. We have applied this technique to analysis of reporter transcripts from a single, integrated copy, or multicopy plasmids. We have evaluated the effect of splice site deletions or the presence or absence of a terminator/cleavage site and demonstrated that both splicing and polyadenylation affect the export of these transcripts from the nucleus to the cytoplasm. Moreover, we show that the exported pre-mRNAs are substrates for nonsense codon-mediated decay through the UPF1 pathway. The work presented here demonstrates that the spatial distribution of transcripts will also be an important component of yeast RNA metabolism.

Base Sequence↗
PubMed1994

Association of poly(A) mRNA with microtubules in cultured neurons.

The structural basis for the synthesis of specific proteins within distinct intraneuronal compartments is unknown. We studied the distribution of poly(A) mRNA within cultured cerebrocortical neurons using high resolution in situ hybridization to identify cytoskeletal components that may anchor mRNA. After 1 day in culture, poly(A) mRNA was distributed throughout all of the initial neurites, including the axon-like process. At 4 days in culture, poly(A) mRNA was distributed throughout the cell body and dendritic processes, but confined to the proximal segment of the axon. Poly(A) mRNA was bound to the cytoskeleton as demonstrated by resistance to detergent extraction. Perturbation of microtubules with colchicine resulted in a major reduction of dendritic poly(A) mRNA; however, this distribution was unaffected by cytochalasin. Ultrastructural in situ hybridization revealed that poly(A) mRNA and associated ribosomes were excluded from tightly bundled microtubules.

Actin Cytoskeleton↗
PubMed1994

Single mRNAs visualized by ultrastructural in situ hybridization are principally localized at actin filament intersections in fibroblasts.

Considerable evidence indicates that mRNA associates with structural filaments in the cell (cytoskeleton). This relationship would be an important mechanism to effect mRNA sorting since specific mRNAs could be sequestered at sites within the cell. In addition, it can provide a mechanism for spatial regulation of mRNA expression. However, the precise structural interactions between mRNA and the cytoskeleton have yet to be defined. An objective of this work was to visualize "individual" poly(A) mRNA molecules in situ by electron microscopy to identify their relationship to individual filaments. Poly(A) RNA and filaments were identified simultaneously using antibodies to detect hybridized probe and filaments or actin-binding proteins. In human fibroblasts, most of the poly(A) mRNA (72%) was localized within 5 nm of orthogonal networks of F-actin filaments. Poly(A) mRNA also colocalized with vimentin filaments (29%) and microtubules (< 10%). The sites of mRNA localization were predominantly at filament intersections. The majority of poly(A) mRNA and polysomes colocalized with the actin crosslinking proteins, filamin, and alpha-actinin, and the elongation factor, EF-1 alpha (actin-binding protein; ABP-50). Evidence that intersections contained single mRNA molecules was provided by using a labeled oligo dT probe to prime the synthesis of cDNA in situ using reverse transcriptase. Both the poly(A) and cis sequences of the same mRNA molecule could then be visualized independently. We propose that the cytoskeletal intersection is a mRNA receptor and serves as a "microdomain" where mRNA is attached and functionally expressed.

Actins↗
PubMed1994

Beta-actin mRNA localization is regulated by signal transduction mechanisms.

Beta-actin mRNA is localized in the leading lamellae of chicken embryo fibroblasts (CEFs) (Lawrence, J., and R. Singer. 1986. Cell. 45:407-415), close to where actin polymerization in the lamellipodia drives cellular motility. During serum starvation beta-actin mRNA becomes diffuse and non-localized. Addition of FCS induces a rapid (within 2-5 min) redistribution of beta-actin mRNA into the leading lamellae. A similar redistribution was seen with PDGF, a fibroblast chemotactic factor. PDGF-induced beta-actin mRNA redistribution was inhibited by the tyrosine kinase inhibitor herbimycin, indicating that this process requires intact tyrosine kinase activity, similar to actin filament polymerization and chemotaxis. Lysophosphatidic acid, which has been shown to rapidly induce actin stress fiber formation (Ridley, A., and A. Hall. 1992. Cell. 790:389-399), also increases peripheral beta-actin mRNA localization within minutes. This suggests that actin polymerization and mRNA localization may be regulated by similar signaling pathways. Additionally, activators or inhibitors of kinase A or C can also delocalize steady-state beta-actin mRNA in cells grown in serum, and can inhibit the serum induction of peripherally localized beta-actin mRNA in serum-starved CEFs. These data show that physiologically relevant extracellular factors operating through a signal transduction pathway can regulate spatial sites of actin protein synthesis, which may in turn affect cellular polarity and motility.

Actins↗
PubMed1994

Localization of HIV RNA in mitochondria of infected cells: potential role in cytopathogenicity.

The intracellular distribution of HIV-1 RNA transcripts in infected cells was studied using in situ hybridization detected by electron microscopy and cellular fractionation. Although viral RNA and core protein could be detected throughout the cytoplasm and nucleus, viral RNA was found in significantly increased amounts in mitochondria relative to the cytoplasm and nucleus. In contrast, cellular poly(A) RNA or viral gag proteins were not increased in the mitochondria. A cell line containing an integrated latent genome that could be induced to express viral RNA after phorbol ester stimulation showed an increase in viral RNA accumulation in mitochondria parallel with the increase in HIV expression levels. Concomitant with HIV expression, there was a decrease in mitochondrial viability. Using immunofluorescent markers to detect probes to HIV RNA transcripts and antibodies to mitochondrial proteins simultaneously in single cells, there was an inverse relationship between the amount of viral RNA and mitochondrial integrity. High levels of viral RNA in mitochondria were found in acutely (but not chronically) infected cells. We propose that HIV RNA import into mitochondria can compromise mitochondrial function.

Cell Line↗
PubMed1994

Sequences responsible for intracellular localization of beta-actin messenger RNA also affect cell phenotype.

We have characterized the structure and function of RNA sequences that direct beta-cytoplasmic actin mRNA to the cell periphery were mapped to two segments of 3'-untranslated region by expression of LacZ/beta-actin chimeric mRNAs in chicken embryo fibroblasts (CEFs). A 54-nt segment, the "RNA zipcode," and a homologous but less active 43-nt segment each localized beta-galactosidase activity to the leading lamellae. This zipcode contains the full activity, and mutations or deletions within it reduce, but do not eliminate, its activity, indicating that several motifs contribute to the activity. Two of these motifs, when multimerized, can regenerate almost full activity. These sequences are highly conserved in evolution, since the human beta-actin zipcode, positioned identically in the 3'UTR localizes equally well in chicken cells. Complementary phosphorothioate oligonucleotides against the zipcode delocalized endogenous beta-actin mRNA, whereas those complementary to the region just outside the zipcode, or sense oligonucleotides, did not. Actin mRNA or protein levels were unaffected by the antisense treatments, but a dramatic change in lamellipodia structure, and actin stress fiber organization was observed using the same antizipcode oligonucleotides which delocalized the mRNA. Hence, discrete 3'UTR sequences direct beta-actin isoform synthesis to the leading lamellae and affect cell morphology, presumably through the actin cytoskeleton.

Actins↗
PubMed1994

Impact of joint impairment on longitudinal disability in elderly persons.

Recent longitudinal data indicating that arthritis is a major contributor to disability in elderly persons are based on self-reported diagnostic information. This longitudinal study included baseline physical examinations of joints of 541 persons over age 60. Previous results from a cross-sectional multivariate model of disability in this sample found that joint impairment (and, its absence, arthritis pain) explained a significant proportion of variance in overall disability. We have retested this model using generalized estimation equations (GEE) analysis to estimate the effect of joint impairment and arthritis pain on baseline and Year 2 disability. Findings indicate that baseline joint impairment contributes substantially to longitudinal disability. If direct measures of baseline joint impairment are unavailable, concurrent self-reported arthritis pain also predicts longitudinal disability well. These findings indicate that longitudinal studies should monitor arthritis pain and that symptomatic arthritis is a risk factor for future disability.

Activities of Daily Living↗
PubMed1993

Isoform-specific 3'-untranslated sequences sort alpha-cardiac and beta-cytoplasmic actin messenger RNAs to different cytoplasmic compartments.

We demonstrate that in differentiating myoblasts, the mRNAs encoding two actin isoforms, beta-cytoplasmic, and alpha-cardiac, can occupy different cytoplasmic compartments within the same cytoplasm. beta-actin mRNA is localized to the leading lamellae and alpha-actin mRNA is associated with a perinuclear compartment. This was revealed by co-hybridizing, in situ, fluorochrome-conjugated oligonucleotide probes specific for each isoform. To address the mechanism of isoform-specific mRNA localization, molecular chimeras were constructed by insertion of actin sequences between the Lac Z coding region and SV-40 3'UTR in a reporter plasmid. These constructs were transiently expressed in a mixed culture of embryonic fibroblasts, myoblasts and myotubes, beta-galactosidase activity within transfectants was revealed by a brief incubation with its substrate (X-gal). Since the blue-insoluble reaction product co-localized with the specific mRNAs expressed from each construct, it was used as a bioassay for mRNA localization. Transfectants were scored as either perinuclear, peripheral or nonlocalized with respect to the distribution of the blue product. The percentage of transfectants within those categories was quantitated as a function of the various constructs. This analysis revealed that for each actin mRNA its 3'UTR is necessary and sufficient to direct reporter transcripts to its appropriate compartment; beta-actin peripheral and alpha-actin perinuclear. In contrast, sequences from the 5'UTR through the coding region of either actin gene did not localize the blue product. Therefore, 3'UTR sequences play a key role in modulating the distribution of actin mRNAs in muscle cells. We propose that the mechanism of mRNA localization facilitates actin isoform sorting in the cytoplasm.

Actins↗