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T Pederson

Publications and source records attributed to T Pederson.

At least 55 records · Page 3Linked to original sources
PubMed1990

Crosslinking of hnRNP proteins to pre-mRNA requires U1 and U2 snRNPs.

Proteins interacting with pre-mRNAs during early stages of spliceosome formation in a HeLa nuclear extract were investigated by photochemical RNA-protein crosslinking. The level of protein crosslinking to a beta-globin pre-mRNA was positively correlated with the presence of an intron. Proteins of 110,000, 59,000 and 39,000 mol. wt. were crosslinked to the beta-globin pre-mRNA, the latter of which was identified as the A1 hnRNP protein. Comparable experiments with an adenovirus pre-mRNA revealed crosslinked proteins of 110,000, 56,000 and 45,000 mol. wt., with the latter identified as belonging to the C group hnRNP proteins. Crosslinking of hnRNP proteins to both the beta-globin and adenovirus pre-mRNAs was eliminated by oligodeoxynucleotide-directed RNase H excision of an internal region (nt 28-42) of U2 RNA, but was not affected by oligo/RNase H cleavage of the 5'-terminal 15 nucleotides of U2 RNA. Cleavage of the 5'-terminal 15 nucleotides of U1 RNA preferentially eliminated crosslinking of the hnRNP A1 protein to both pre-mRNAs. The requirement of intact U1 snRNP for A1 protein crosslinking was further demonstrated by the fact that although micrococcal nuclease-treated extracts did not support crosslinking of A1 hnRNP protein to beta-globin pre-mRNA, crosslinking was restored by addition of a U1 snRNP-enriched fraction.

Base Sequence↗
PubMed1990

An intron-containing Schizosaccharomyces pombe U6 RNA gene can be transcribed by human RNA polymerase III.

A Schizosaccharomyces pombe U6 small nuclear RNA gene containing an intron has been described. We find that the S. pombe U6 gene is transcribed in a human (HeLa) cell S100 extract with an alpha-amanitin sensitivity characteristic of RNA polymerase III. The S. pombe U6 gene is also transcribed after transfection into human cells. The transcription of vertebrate U6 RNA genes by RNA polymerase III does not require intragenic control elements. The intron of the S. pombe U6 gene disrupts a "box A"-like intragenic sequence that is typically an RNA polymerase III transcription control element. This, together with the transcription of the S. pombe U6 gene by human RNA polymerase III, suggests that it is recognized by human U6 gene-specific transcription machinery.

Amanitins↗
PubMed1990

RNA processing and ribonucleoprotein assembly studied in vivo by RNA transfection.

We present a method for studying RNA processing and ribonucleoprotein assembly in vivo, by using RNA synthesized in vitro. SP6-transcribed 32P-labeled U2 small nuclear RNA precursor molecules were introduced into cultured human 293 cells by calcium phosphate-mediated uptake, as in standard DNA transfection experiments. RNase protection mapping demonstrated that the introduced pre-U2 RNA underwent accurate 3' end processing. The introduced U2 RNA was assembled into ribonucleoprotein particles that reacted with an antibody specific for proteins known to be associated with the U2 small nuclear ribonucleoprotein particle. The 3' end-processed, ribonucleoprotein-assembled U2 RNA accumulated in the nuclear fraction. When pre-U2 RNA with a 7-methylguanosine group at the 5' end was introduced into cells, it underwent conversion to a 2,2,7-trimethylguanosine cap structure, a characteristic feature of the U-small nuclear RNAs. Pre-U2 RNA introduced with an adenosine cap (Ap-ppG) also underwent processing, small nuclear ribonucleoprotein assembly, and nuclear accumulation, establishing that a methylated guanosine cap structure is not required for these steps in U2 small nuclear ribonucleoprotein biosynthesis. Beyond its demonstrated usefulness in the study of small nuclear ribonucleoprotein biosynthesis, RNA transfection may be of general applicability to the investigation of eukaryotic RNA processing in vivo and may also offer opportunities for introducing therapeutically targeted RNAs (ribozymes or antisense RNA) into cells.

Base Sequence↗
PubMed1990

Site-specific excision from RNA by RNase H and mixed-phosphate-backbone oligodeoxynucleotides.

Oligodeoxynucleotides containing phosphodiester or modified internucleoside linkages were investigated with respect to their ability to be acted on by ribonuclease H activities present in a HeLa cell nuclear extract after hybridization with complementary sequences in RNA. Oligodeoxynucleotides complementary to nucleotides 2-14 of human U1 small nuclear RNA were investigated. Extensive cleavage of U1 RNA was observed with the unmodified oligodeoxynucleotide and with the phosphorothioate analogue but not with U1-complementary oligodeoxynucleotides containing methylphosphonate, phosphoro-N-morpholidate, or phosphoro-N-butylamidate internucleoside linkages. Additional experiments using a 514-nucleotide-long RNA substrate demonstrated the capacity of complementary phosphodiester- and phosphorothioate-linked oligodeoxynucleotides (but not ones containing methylphosphonate, phosphoro-N-morpholidate, or phosphoro-N-butylamidate linkages) to serve as RNase H targets when hybridized to an internal RNA site. Detailed comparisons revealed phosphodiester-linked oligodeoxynucleotides to be more efficient than the comparable phosphorothioate-linked oligomers with respect to RNase H action. Various pentadecamer oligodeoxynucleotides complementary to the 514-nucleotide-long test RNA and containing 2-6 consecutive phosphodiester- or phosphorothioate-linked nucleotides flanked by RNase H-resistant methylphosphonate linkages afforded precise "site-directed" RNase H excision within the DNA.RNA hybrid. These results serve to assort modified oligodeoxynucleotide-containing hybrids into RNase H-sensitive and -resistant classes and also provide clues as to how RNase H makes contact with the DNA strand in a DNA.RNA hybrid.

Base Sequence↗
PubMed1989

[Monitoring routines at Danish anesthesia departments].

By means of a questionnaire investigation, 84 Danish anaesthetic departments were questioned in January 1987 about their monitoring routines during anaesthesia and recovery. 100% replied. The investigation revealed that measurement of the blood pressure in adults was the routine in all of the departments. Patients were monitored routinely with EGG in 62 departments where minor surgical interventions were concerned while, in major surgical intervention, ECG monitoring was the routine in 91% of the departments. Patients were observed postoperatively in the recovery room or intensive care unit in 51% of the departments. The questionnaire investigation revealed great variation in the monitoring and supervision routines in the anaesthetic departments in Denmark. Hitherto, it has not been possible to demonstrate that monitoring of the functions of vital organs in operation patients can reduce the morbidity and mortality connected with the anaesthesia. It is concluded that it would be desirable to carry out clinically controlled investigations to illustrate whether monitoring equipment can reduce the morbidity connected with anaesthesia and to develop a Danish recommendation concerning peroperative monitoring and postoperative observation.

Anesthesia Department, Hospital↗
PubMed1989

Transcription of a human U6 small nuclear RNA gene in vivo withstands deletion of intragenic sequences but not of an upstream TATATA box.

Most eukaryotic genes transcribed by RNA polymerase III contain internal control regions. U6 small nuclear RNA genes are transcribed by RNA polymerase III but are unusual in that, at least in vitro, their expression does not require intragenic sequences. Here we show that this is true as well in vivo. A human U6 gene devoid of all but the first 6 and last 10 base-pairs was expressed efficiently after transfection into human 293 cells. We also report data extending the previous identification of 5' flanking sequences important for human U6 gene transcription. Deletion-substitution of a 10 base-pair upstream sequence encompassing the TATATA element (-29 to -24) abolished U6 transcription. A double point mutation in the middle of this element (TATATA-TAGCTA) reduced U6 transcription but not to the extent brought about by TATATA deletion-substitution. These results establish that, in vivo, transcription of human U6 small nuclear RNA is independent of intragenic sequences between nucleotides 6 and 98, and requires the upstream TATATA box.

DNA Mutational Analysis↗
PubMed1989

U2 small nuclear RNP assembly in vitro.

Incubation of a SP6-transcribed human U2 RNA precursor molecule in a HeLa cell S100 fraction resulted in the formation of ribonucleoprotein complexes. In the presence of ATP, the particles that assembled had several properties of native U2 snRNP, including resistance to dissociation in Cs2SO4 gradients, their buoyant density, and pattern of digestion by micrococcal nuclease. These particles also reacted with Sm monoclonal antibody and a human autoantibody with specificity for the U2 snRNP-specific proteins A' and B", but not with antibodies for U1 snRNP-specific proteins. In contrast, the particles that formed in the absence of ATP did not have these properties. ATP analogs with non-hydrolyzable beta-gamma bonds did not substitute for ATP in U2 snRNP assembly. Additional experiments with a mutant U2 RNA confirmed that nucleotides 154-167 of U2 RNA are required for binding of the U2 snRNP-specific proteins but not of the "Sm" core proteins. Pseudouridine formation, a major post-transcriptional modification of U2 RNA, was enhanced under assembly permissive conditions.

Adenosine Triphosphate↗
PubMed1989

U1 small nuclear ribonucleoprotein particle-specific proteins interact with the first and second stem-loops of U1 RNA, with the A protein binding directly to the RNA independently of the 70K and Sm proteins.

The U1 small nuclear ribonucleoprotein particle (U1 snRNP), a cofactor in pre-mRNA splicing, contains three proteins, termed 70K, A, and C, that are not present in the other spliceosome-associated snRNPs. We studied the binding of the A and C proteins to U1 RNA, using a U1 snRNP reconstitution system and an antibody-induced nuclease protection technique. Antibodies that reacted with the A and C proteins induced nuclease protection of the first two stem-loops of U1 RNA in reconstituted U1 snRNP. Detailed analysis of the antibody-induced nuclease protection patterns indicated the existence of relatively long-range protein-protein interactions in the U1 snRNP, with the 5' end of U1 RNA and its associated specific proteins interacting with proteins bound to the Sm domain near the 3' end. UV cross-linking experiments in conjunction with an A-protein-specific antibody demonstrated that the A protein bound directly to the U1 RNA rather than assembling in the U1 snRNP exclusively via protein-protein interactions. This conclusion was supported by additional experiments revealing that the A protein could bind to U1 RNA in the absence of bound 70K and Sm core proteins.

Autoantibodies↗
PubMed1988

The Mr 70,000 protein of the U1 small nuclear ribonucleoprotein particle binds to the 5' stem-loop of U1 RNA and interacts with Sm domain proteins.

The U1 small nuclear ribonucleoprotein (snRNP) particle, a cofactor in mRNA splicing, contains nine proteins, six of which are also present in other U snRNPs and three of which are specific to the U1 snRNP. Here we have used a reconstituted human U1 snRNP together with snRNP monoclonal antibodies to define the RNA binding sites of one of the U1 snRNP-specific proteins. When Sm monoclonal antibody (specific for the B', B, and D proteins of U snRNPs) was bound to U1 snRNPs prior to micrococcal nuclease digestion, the same approximately equal to 24 nucleotide fragment of U1 RNA (corresponding to nucleotides 120-143 and termed the "Sm domain") was protected as when no antibody was bound prior to digestion. In contrast, when RNP monoclonal antibody, which reacts with the U1 snRNP-specific Mr 70,000 protein, was bound, additional U1 RNA regions were protected against nuclease digestion. This phenomenon, which we term "antibody-mediated nuclease protection," was exploited to map the position of the Mr 70,000 protein to stem-loop I of U1 RNA. However, there were also sites of Mr 70,000 protein interaction with more 3'-ward regions of U1 RNA, particularly the Sm domain. This indicates that in the three-dimensional structure of the U1 snRNP, the RNP and Sm antigens are in contact with each other. The proximity of the Mr 70,000 protein's RNA binding site (stem-loop I) to the functionally important 5' end of U1 RNA suggests that this protein may be involved in the recognition of, or stabilization of base pairing with, pre-mRNA 5' splice sites.

Antibodies, Monoclonal↗
PubMed1988

Upstream elements required for efficient transcription of a human U6 RNA gene resemble those of U1 and U2 genes even though a different polymerase is used.

U6 small nuclear RNA is transcribed by a different polymerase than U1-U5 RNAs, likely to be RNA polymerase III. Transcription from human U6 gene deletion-substitution templates in a HeLa S100 extract delineated the 5' border of a control element lying between 67 and 43 bp upstream from the initiation site. This region matches the location of, and shows considerable sequence similarity with, the proximal control element of U1 and U2 RNA genes, which are transcribed by RNA polymerase II. Transfection of human 293 cells with 5'-flanking deletion-substitution mutants of a U6 maxigene revealed a dominant control element between 245 and 149 bp upstream of the transcription start site. An octamer motif was found in this region in an inverted orientation relative to that of the human U1 and U2 RNA gene enhancers but in the same orientation as a human U4 RNA gene, the transcript of which functions together with U6 RNA in a single small nuclear ribonucleoprotein (snRNP) particle. The human U2 gene enhancer joined to the U6 maxigene was able to functionally replace the U6 distal control element(s).

Base Sequence↗
PubMed1987

Reconstitution of the U1 small nuclear ribonucleoprotein particle.

Although the U1 small nuclear ribonucleoprotein particle (snRNP) was the first mRNA-splicing cofactor to be identified, the manner in which it functions in splicing is not precisely understood. Among the information required to understand how U1 snRNP participates in splicing, it will be necessary to know its structure. Here we describe the in vitro reconstitution of a particle that possesses the properties of native U1 snRNP. 32P-labeled U1 RNA was transcribed from an SP6 promoter-human U1 gene clone and incubated in a HeLa S100 fraction. A U1 particle formed which displayed the same sedimentation coefficient (approximately 10S) and buoyant density (1.40 g/cm3) as native U1 snRNP. The latter value reflects the ability to withstand isopycnic banding in Cs2SO4 without prior fixation, a property shared by native U1 snRNP. The reconstituted U1 particle reacted with both the Sm and RNP monoclonal antibodies, showing that these two classes of snRNP proteins were present. Moreover, the reconstituted U1 snRNP particle was found to display the characteristic Mg2+ switch of nuclease sensitivity previously described for native U1 snRNP: an open, nuclease-sensitive conformation at a low Mg2+ concentration (3 mM) and a more compact, nuclease-resistant organization at a higher concentration (15 mM). The majority of the U1 RNA in the reconstituted particle did not contain hypermethylated caps, pseudouridine, or ribose 2-O-methylation, showing that these enigmatic posttranscriptional modifications are not essential for reconstitution of the U1 snRNP particle. The extreme 3' end (18 nucleotides) of U1 RNA was required for reconstitution, but loop II (nucleotides 64 to 77) was not. Interestingly, the 5' end (15 nucleotides) of U1 RNA that recognizes pre-mRNA 5' splice sites was not required for U1 snRNP reconstruction.

Humans↗
PubMed1987

Accurate and efficient 3' processing of U2 small nuclear RNA precursor in a fractionated cytoplasmic extract.

The small nuclear RNAs U1, U2, U4, and U5 are cofactors in mRNA splicing and, like the pre-mRNAs with which they interact, are transcribed by RNA polymerase II. Also like mRNAs, mature U1 and U2 RNAs are generated by 3' processing of their primary transcripts. In this study we have investigated the in vitro processing of an SP6-transcribed human U2 RNA precursor, the 3' end of which matches that of authentic human U2 RNA precursor molecules. Although the SP6-U2 RNA precursor was efficiently processed in an ammonium sulfate-fractionated HeLa cytoplasmic S100 extract, the product RNA was unstable. Further purification of the processing activity on glycerol gradients resolved a 7S activity that nonspecifically cleaved all RNAs tested and a 15S activity that efficiently processed the 3' end of pre-U2 RNA. The 15S activity did not process the 3' end of a tRNA precursor molecule. As demonstrated by RNase protection, the processed 3' end of the SP6-U2 RNA maps to the same nucleotides as does mature HeLa U2 RNA.

Cytoplasm↗
PubMed1987

Evidence for an association between U1 RNA and interspersed repeat single-copy RNAs in the cytoplasm of sea urchin eggs.

Psoralen crosslinking of RNA-RNA intermolecular duplexes in sea urchin egg extracts reveals that some maternal poly(A)+ RNA molecules are complexed with U1 RNA, a cofactor in somatic nuclear pre-mRNA splicing. Reaction of egg extracts with a monoclonal antibody specific for U1 snRNP selects, in addition to U1, RNAs that contain repeated sequences interspersed with single-copy elements. Antibody-selection experiments with nucleate and anucleate egg halves demonstrate that most of the U1 RNA-interspersed RNA complexes are cytoplasmic, as is the egg's store of total U1 snRNP. These results raise the possibility that maternal interspersed RNAs include unprocessed pre-messenger RNA molecules in arrested complexes with splicing cofactors.

Animals↗
PubMed1986

Identification of proteins that bind tightly to pre-mRNA during in vitro splicing.

Incubation of a human beta-globin pre-mRNA in a HeLa cell nuclear extract under conditions permissive for efficient splicing resulted in the assembly of the RNA into ribonucleoprotein (RNP) complexes. This RNP formation occurred largely within the characteristic lag period that precedes splicing. Two classes of RNP were detected by the criterion of their stability in Cs2SO4 gradients. One was unstable and contained mainly aberrant RNA cleavage products. The other class of RNP complexes comprised 50-85% of the beta-globin RNA, formed only under splicing-permissive conditions, was stable in Cs2SO4 gradients, and contained both unspliced pre-mRNA molecules and the lariat intron 1-exon 2 splicing intermediate. This latter class of RNP complexes banded at approximately equal to 1.30 g/cm3, a density very similar to that of native heterogeneous nuclear RNP particles that contain pre-mRNA. RNA-protein crosslinking revealed major proteins of Mr approximately equal to 38,000 and 41,000 in the stable class of RNP. The use of antibodies specific for heterogeneous nuclear RNP core proteins and for small nuclear RNA-associated proteins, in conjunction with [32P]RNA-protein crosslinking, revealed polypeptides having the molecular weights of both sets of antigens. These results show that both heterogeneous nuclear RNP particle core proteins and small nuclear RNA-associated proteins bind tightly to pre-mRNA during splicing in vitro.

Autoantibodies↗
PubMed1986

U6 small nuclear RNA is transcribed by RNA polymerase III.

A DNA fragment homologous to U6 small nuclear RNA was isolated from a human genomic library and sequenced. The immediate 5'-flanking region of the U6 DNA clone had significant homology with a potential mouse U6 gene, including a "TATA box" at a position 26-29 nucleotides upstream from the transcription start site. Although this sequence element is characteristic of RNA polymerase II promoters, the U6 gene also contained a polymerase III "box A" intragenic control region and a typical run of five thymines at the 3' terminus (noncoding strand). The human U6 DNA clone was accurately transcribed in a HeLa cell S100 extract lacking polymerase II activity. U6 RNA transcription in the S100 extract was resistant to alpha-amanitin at 1 microgram/ml but was completely inhibited at 200 micrograms/ml. A comparison of fingerprints of the in vitro transcript and of U6 RNA synthesized in vivo revealed sequence congruence. U6 RNA synthesis in isolated HeLa cell nuclei also displayed low sensitivity to alpha-amanitin, in contrast to U1 and U2 RNA transcription, which was inhibited greater than 90% at 1 microgram/ml. In addition, U6 RNA synthesized in isolated nuclei was efficiently immunoprecipitated by an antibody against the La antigen, a protein known to bind most other RNA polymerase III transcripts. These results establish that, in contrast to the polymerase II-directed transcription of mammalian genes for U1-U5 small nuclear RNAs, human U6 RNA is transcribed by RNA polymerase III.

Amanitins↗
PubMed1985

Ribonucleoprotein organization of eukaryotic RNA. XXXII. U2 small nuclear RNA precursors and their accurate 3' processing in vitro as ribonucleoprotein particles.

Biosynthetic precursors of U2 small nuclear RNA have been identified in cultured human cells by hybrid-selection of pulse-labeled RNA with cloned U2 DNA. These precursor molecules are one to approximately 16 nucleotides longer than mature U2 RNA and contain 2,2,7-trimethylguanosine "caps". The U2 RNA precursors are associated with proteins that react with a monoclonal antibody for antigens characteristic of small nuclear ribonucleoprotein particles. Like previously described precursors of U1 and U4 small nuclear RNAs, the pre-U2 RNAs are recovered in cytoplasmic fractions, although it is not known if this is their location in vivo. The precursors are processed to mature-size U2 RNA when cytoplasmic extracts are incubated in vitro at 37 degrees C. Mg2+ is required but ATP is not. The ribonucleoprotein structure of the pre-U2 RNA is maintained during the processing reaction in vitro, as are the 2,2,7-trimethylguanosine caps. The ribonucleoprotein organization is of major importance, as exogenous, protein-free U2 RNA precursors are degraded rapidly in the in vitro system. Two lines of evidence indicate that the conversion of U2 precursors to mature-size U2 RNA involves a 3' processing reaction. First, the reaction is unaffected by a large excess of mature U2 small nuclear RNP, whose 5' trimethylguanosine caps would be expected to compete for a 5' processing activity. Second, when pre-U2 RNA precursors are first stoichiometrically decorated with an antibody specific for 2,2,7-trimethylguanosine, the extent of subsequent processing in vitro is unaffected. These results provide the first demonstration of a eukaryotic RNA processing reaction in vitro occurring within a ribonucleoprotein particle.

Autoantibodies↗
PubMed1985

cDNA cloning of a human autoimmune nuclear ribonucleoprotein antigen.

Sera from patients with systemic lupus erythematosus and other autoimmune disorders contain antibodies against nuclear proteins. One such autoantibody system, known as Sm, reacts with antigens associated with small nuclear RNA molecules. In this paper we report the use of Sm autoantibodies to isolate a cDNA clone for the mRNA of one of these nuclear antigens. A HeLa cell cDNA library was screened by message selection followed by autoantibody reaction of cell-free translation products. This led to the identification of a cDNA clone, p281, containing sequences complementary to mRNA for an Sm autoantibody-reactive, 11,000 Mr protein. This cloned Sm antigen comigrated with the small nuclear RNA-associated protein known as "E" and reacted with four out of four Sm autoantibodies that precipitate E protein from total mRNA translation products. RNA gel blot hybridization with clone p281 DNA revealed a poly(A)+ mRNA of approximately equal to 600 nucleotides in human and marmoset (New World primate) cells. Southern blot hybridization of HeLa cell and human lymphocyte DNA indicated the presence of 6-10 copies of p281-homologous sequences. Similar copy numbers were observed with genomic DNA from baboon, cat, and mouse, indicating that the Sm antigen mRNA sequence represented in p281 is conserved across three classes of the Mammalia (primates, carnivores, and rodents). However, no cross-hybridization of p281 was observed with frog or Drosophila DNA. In light of existing evidence that the mammalian Sm antigen E is a weaker autoantigen than other small nuclear RNA-associated proteins, these results suggest a possible correlation between a protein's capacity to serve as an autoantigen during breakdown of the host's immunological tolerance and its extent of evolutionary conservation, whereas the inverse relationship applies to conventional immunity. We suspect, as have others, that this is a clue to the mechanism of autoimmunity.

Antigens↗