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T Pederson

Publications and source records attributed to T Pederson.

At least 19 recordsLinked to original sources
PubMed1999

A human U2 RNA mutant stalled in 3' end processing is impaired in nuclear import.

The biosynthesis of U1, U2, U4 and U5 spliceosomal small nuclear RNAs (snRNAs) involves the nuclear export of precursor molecules extended at their 3' ends, followed by a cytoplasmic phase during which the pre-snRNAs assemble into ribonucleoprotein particles and undergo hypermethylation of their 5' caps and 3' end processing prior to nuclear import. Previous studies have demonstrated that the assembly of pre-snRNAs into ribonucleoprotein particles containing the Sm core proteins is essential for nuclear import in mammalian cells but that 5' cap hypermethylation is not. In the present investigation we have asked whether or not 3' end processing is required for nuclear import of U2 RNA. We designed human pre-U2 RNAs that carried modified 3' tails, and identified one that was stalled (or greatly slowed) in 3' end processing, leading to its accumulation in the cytoplasm of human cells. Nonetheless, this 3' processing arrested pre-U2 RNA molecule was found to undergo cytoplasmic assembly into Sm protein-containing complexes to the same extent as normal pre-U2 RNA. The Sm protein-associated, unprocessed mutant pre-U2 RNA was not observed in the nuclear fraction. Using an assay based on suppression of a genetically blocked SV40 pre-mRNA splicing pathway, we found that the 3' processing deficient U2 RNA was significantly reduced in its ability to rescue splicing, consistent with its impaired nuclear import.

3' Untranslated Regions↗
PubMed1998

The plurifunctional nucleolus.

The nucleolus of eukaryotic cells was first described in the early 19th century and was discovered in the 1960s to be the seat of ribosome synthesis. Although rRNA transcription, rRNA processing and ribosome assembly have been clearly established as major functions of the nucleolus, recent studies suggest that the nucleolus participates in many other aspects of gene expression as well. Thus, the nucleolus has been implicated in the processing or nuclear export of certain mRNAs. In addition, new results indicate that biosyntheses of signal recognition particle RNA and telomerase RNA involve a nucleolar stage and that the nucleolus is also involved in processing of U6 RNA, one of the spliceosomal small nuclear RNAs. Interestingly, these three nucleolus-associated small nuclear RNAs (signal recognition particle RNA, telomerase RNA and U6 RNA) are components of catalytic ribonucleoprotein machines. Finally, recent work has also suggested that some transfer RNA precursors are processed in the nucleolus. The nucleolus may have evolutionarily descended from a proto-eukaryotic minimal genome that was spatially linked to vicinal RNA processing and ribonucleoprotein assembly events involved in gene read-out. The nucleolus of today's eukaryotes, now surrounded by the chromatin of over 2 billion years of genome expansion, may still perform these ancient functions, in addition to ribosome biosynthesis. The plurifunctional nucleolus concept has a strong footing in contemporary data and adds a new perspective to our current picture of the spatial-functional design of the cell nucleus.

Cell Nucleolus↗
PubMed1998

Localization of signal recognition particle RNA in the nucleolus of mammalian cells.

The signal recognition particle (SRP) of eukaryotic cells is a cytoplasmic ribonucleoprotein machine that arrests the translational elongation of nascent secretory and membrane proteins and facilitates their transport into the endoplasmic reticulum. The spatial pathway of SRP RNA processing and ribonucleoprotein assembly in the cell is not known. In the present investigation, microinjection of fluorescently tagged SRP RNA into the nucleus of mammalian cells was used to examine its intranuclear sites of localization. Microinjection of SRP RNA into the nuclei of normal rat kidney (NRK) epithelial cells maintained at 37 degreesC on the microscope stage resulted in a very rapid initial localization in nucleoli, followed by a progressive decline of nucleolar signal and an increase of fluorescence at discrete sites in the cytoplasm. Nuclear microinjection of a molecule corresponding to a major portion of the Alu domain of SRP RNA revealed a pattern of rapid nucleolar localization followed by cytoplasmic appearance of signal that was similar to the results obtained with full-length SRP RNA. In contrast, a molecule corresponding to the S domain of SRP RNA did not display nucleolar localization to the extent observed with full-length SRP RNA. An SRP RNA molecule lacking helix 6 of the S domain displayed normal nucleolar localization, whereas one lacking helix 8 of the S domain did not. These results, obtained by direct, real-time observation of fluorescent RNA molecules inside the nucleus of living mammalian cells, suggest that the processing of SRP RNA or its ribonucleoprotein assembly into the SRP involves a nucleolar phase.

Animals↗
PubMed1998

Intranuclear diffusion and hybridization state of oligonucleotides measured by fluorescence correlation spectroscopy in living cells.

Fluorescein-labeled oligodeoxynucleotides (oligos) were introduced into cultured rat myoblasts, and their molecular movements inside the nucleus were studied by fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP). FCS revealed that a large fraction of both intranuclear oligo(dT) (43%) and oligo(dA) (77%) moves rapidly with a diffusion coefficient of 4 x 10(-7) cm2/s. Interestingly, this rate of intranuclear oligo movement is similar to their diffusion rates measured in aqueous solution. In addition, we detected a large fraction (45%) of the intranuclear oligo(dT), but not oligo(dA), diffusing at slower rates (</=1 x 10(-7) cm2/s). The amount of this slower-moving oligo(dT) was greatly reduced if the oligo(dT) was prehybridized in solution with (unlabeled) oligo(dA) prior to introduction to cells, presumably because the oligo(dT) was then unavailable for subsequent hybridization to endogenous poly(A) RNA. The FCS-measured diffusion rate for much of the slower oligo(dT) population approximated the diffusion rate in aqueous solution of oligo(dT) hybridized to a large polyadenylated RNA (1.0 x 10(-7) cm2/s). Moreover, this intranuclear movement rate falls within the range of calculated diffusion rates for an average-sized heterogeneous nuclear ribonucleoprotein particle in aqueous solution. A subfraction of oligo(dT) (15%) moved over 10-fold more slowly, suggesting it was bound to very large macromolecular complexes. Average diffusion coefficients obtained from FRAP experiments were in agreement with the FCS data. These results demonstrate that oligos can move about within the nucleus at rates comparable to those in aqueous solution and further suggest that this is true for large ribonucleoprotein complexes as well.

Animals↗
PubMed1998

Thinking about a nuclear matrix.

The possible existence in eukaryotic cells of an internal, non-chromatin nuclear structural framework that facilitates gene readout as a set of spatially concerted reactions has become a popular but controversial theater of investigation. This article endeavors to present a circumspect review of the nuclear matrix concept as we presently know it, framed around two contrasting hypotheses: (1) that an internal nuclear framework actively enhances gene expression (in much the same way the cytoskeleton mediates cell locomotion, mitosis and intracellular vesicular traffic) versus (2) that the interphase chromosomes have fixed, inherited positions and that the DNA replication, transcripton and RNA processing machinery diffusionally arrives at sites of gene readout, with some aspects of nuclear structure thus being more a result than a cause of gene expression. On balance, the available information suggests that interactions among various gene expression machines may contribute to isolated nuclear matrix preparations. Some components of isolated nuclear matrix preparations may also reflect induced or reconfigured protein-protein associations. The protein characterization and ultrastructural analysis of the isolated nuclear matrix has advanced significantly in recent years, although controversies remain. Important new clues are now coming in from promising contemporary lines of research that report on nuclear structure in living cells.

Cell Nucleus↗
PubMed1998

A 7-methylguanosine cap commits U3 and U8 small nuclear RNAs to the nucleolar localization pathway.

U3 and U8 small nucleolar RNAs (snRNAs) participate in pre-rRNA processing. Like the U1, U2, U4 and U5 major spliceosomal snRNAs, U3 and U8 RNAs are transcribed by RNA polymerase II and their initial 7-methylguanosine (m7G) 5' cap structures subsequently become converted to 2,2,7-trimethylguanosine. However, unlike the polymerase II transcribed spliceosomal snRNAs, which are exported to the cytoplasm for cap hypermethylation, U3 and U8 RNAs undergo cap hypermethylation within the nucleus. Human U3 and U8 RNAs with various cap structures were generated by in vitro transcription, fluorescently labeled and microinjected into nuclei of normal rat kidney (NRK) epithelial cells. When U3 and U8 RNAs containing a m7G cap were microinjected they became extensively localized in nucleoli. U3 and U8 RNAs containing alternative cap structures did not localize in nucleoli nor did U3 or U8 RNAs containing triphosphate 5'-termini. The nucleolar localization of m7G-capped U3 RNA was competed by co-microinjection into the nucleus of a 100-fold molar excess of dinucleotide m7GpppG but not by a 100-fold excess of ApppG dinucleotide. Although it was obviously not possible to assess formation of di- and trimethylguanosine caps on the microinjected U3 and U8 RNAs in these single cell experiments, these results indicate that the initial presence of a m7G cap on U3 and U8 RNAs, most likely together with internal sequence elements, commits these transcripts to the nucleolar localization pathway and point to diverse roles of the m7G cap in the intracellular traffic of various RNAs transcribed by RNA polymerase II.

Animals↗
PubMed1997

RNA-dependent phosphorylation of a nuclear RNA binding protein.

The human C1 heterogeneous nuclear ribonucleoprotein particle protein (hnRNP protein) undergoes a cycle of phosphorylation-dephosphorylation in HeLa cell nuclear extracts that modulates the binding of this protein to pre-mRNA. We now report that hyperphosphorylation of the C1 hnRNP protein is mediated by a kinase activity in nuclear extracts that is RNA-dependent. Although the basal phosphorylation of the C1 hnRNP protein in nuclear extracts reflects a casein kinase II-type activity, its RNA-dependent hyperphosphorylation appears to be mediated by a different kinase. This is indicated by the unresponsiveness of the RNA-stimulated hyperphosphorylation to casein kinase II inhibitors, and the distinct glycerol gradient sedimentation profiles of the basal versus RNA-stimulated C1 hnRNP protein phosphorylation activities from nuclear extracts. RNA-dependent phosphorylation was observed both for a histidine-tagged recombinant human C1 hnRNP protein added to nuclear extracts and also for the endogenous C1 hnRNP protein. Additional results rule out protein kinase A, protein kinase C, calmodulin-dependent protein kinase II, and double-stranded RNA-activated protein kinase as the enzymes responsible for the RNA-dependent hyperphosphorylation of the C1 hnRNP protein. These results reveal the existence in nuclear extracts of an RNA-dependent protein kinase activity that hyperphosphorylates a known pre-mRNA binding protein, and define an additional element to be integrated into the current picture of how nuclear proteins are regulated by phosphorylation.

Casein Kinase II↗
PubMed1997

3' processing of human pre-U2 small nuclear RNA: a base-pairing interaction between the 3' extension of the precursor and an internal region.

The spliceosomal small nuclear RNAs U1, U2, U4, and U5 are transcribed by RNA polymerase II as precursors with extensions at their 3' ends. The 3' processing of these pre-snRNAs is not understood in detail. Two pathways of pre-U2 RNA 3' processing in vitro were revealed in the present investigation by using a series of human wild-type and mutant pre-U2 RNAs. Substrates with wild-type 3' ends were initially shortened by three or four nucleotides (which is the first step in vivo), and the correct mature 3' end was then rapidly generated. In contrast, certain mutant pre-U2 RNAs displayed an aberrant 3' processing pathway typified by the persistence of intermediates representing cleavage at each internucleoside bond in the precursor 3' extension. Comparison of the wild-type and mutant pre-U2 RNAs revealed a potential base-pairing interaction between nucleotides in the precursor 3' extension and a sequence located between the Sm domain and stem-loop III of U2 RNA. Substrate processing competition experiments using a highly purified pre-U2 RNA 3' processing activity suggested that only RNAs capable of this base-pairing interaction had high affinity for the pre-U2 RNA 3' processing enzyme. The importance of this postulated base-pairing interaction between the precursor 3' extension and the internal region between the Sm domain and stem-loop III was confirmed by the results obtained with a compensatory substitution that restores the base pairing, which displayed the normal 3' processing reaction. These results implicate a precursor-specific base-paired structure involving sequences on both sides of the mature cleavage site in the 3' processing of human U2 RNA.

Base Composition↗
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

The C-group heterogeneous nuclear ribonucleoprotein proteins bind to the 5' stem-loop of the U2 small nuclear ribonucleoprotein particle.

The C-group heterogeneous nuclear ribonucleoprotein (hnRNP) proteins bind to nascent pre-messenger RNA. In vitro studies have indicated that the C hnRNP proteins bind particularly strongly to the intron polypyrimidine tract of pre-mRNA and may be important for pre-mRNA splicing. In addition, there is evidence that the interaction of the C hnRNP proteins with pre-mRNA is facilitated by the U1 and U2 small nuclear RNPs (snRNPs). In the present study, we have uncovered another feature of the C hnRNP proteins that may provide a unifying framework for these previous observations; the C hnRNP proteins bind to the 5' stem-loop of the U2 snRNP. This was detected by incubating human 32P-labeled U2 snRNP in micrococcal nuclease-treated HeLa nuclear extracts, followed by UV-mediated protein-RNA cross-linking, which revealed that C hnRNP proteins were cross-linked to 32P-nucleotides in the U2 snRNP. In similar experiments, no cross-linking of C hnRNP proteins to 32P-labeled U1 or U4 snRNPs was observed. The observed cross-linking of C hnRNP proteins to U2 snRNP was efficiently competed by excess U2 RNA and by poly(U) but not by poly(A). No competition was observed with an RNA molecule comprising U2 nucleotides 105-189, indicating that the C hnRNP protein interactive regions of U2 RNA reside solely in the 5' half of the molecule. Oligodeoxynucleotide-mediated RNase H cleavage experiments revealed that a 5' region of U2 RNA including nucleotides 15-28 is essential for the observed C hnRNP protein cross-linking. C hnRNP protein cross-linking to U2 snRNP was efficiently competed by a mini-RNA corresponding to the first 29 nucleotides of U2 RNA, whereas no competition was observed with a variant of this mini-RNA in which the UUUU loop of stem-loop I was mutationally configured into a single-stranded RNA by replacing the stem with non-pairing nucleotides. Competition experiments with another mutant mini-U2 RNA in which the UUUU loop was replaced by AAAA indicated that both the UUUU loop and the stem are important for C hnRNP protein cross-linking, a finding consistent with other recent data on the RNA sequence specificity of C hnRNP protein binding.

Binding, Competitive↗
PubMed1996

A discrete 3' region of U6 small nuclear RNA modulates the phosphorylation cycle of the C1 heterogeneous nuclear ribonucleoprotein particle protein.

The C heterogeneous ribonucleoprotein particle (hnRNP) protein bind to nascent pre-mRNA and may participate in assembly of the early prespliceosome. Ser/Thr phosphorylation of the C1 hnRNP protein in HeLa nuclear extracts regulates its binding to pre-mRNA (S. H. Mayrand, P. Dwen, and T. Pederson, Proc. Natl. Acad. Sci. USA 90:7764-7768, 1993). We have now further investigated the phosphorylation cycle of the C1 hnRNP protein, with emphasis on its regulation. Pretreatment of nuclear extracts with micrococcal nuclease eliminated the phosphorylation of C1 hnRNP protein, but pretreatment with DNase did not, suggesting a dependence on RNA. Oligodeoxynucleotide-targeted RNase H cleavage of U1, U2, and U4 small nuclear RNAs did not affect the phosphorylation of C1 hnRNP protein. However, cleavage of nucleotides 78 to 95, but not other regions, of U6 small nuclear RNA resulted in an inhibition of the dephosphorylation step of the C1 hnRNP protein phosphorylation cycle. This inhibition was as pronounced as that seen with the serine/threonine protein phosphatase inhibitor okadaic acid. C1 hnRNP protein dephosphorylation could be completely restored by the addition of intact U6 RNA. Add-back experiments with mutant RNAs further delineated the minimal region essential for C1 protein dephosphorylation as residing in nucleotides 85 to 92 of U6 RNA. These results illuminate a hitherto unanticipated function of U6 RNA: the modulation of a phosphorylation-dephosphorylation cycle of C1 hnRNP protein that influences the binding affinity of this protein for pre-mRNA. This newly revealed function of U6 RNA is likely to play a very early role in the prespliceosome assembly pathway, prior to U6 RNA's entry into the mature spliceosome's active center.

Base Sequence↗
PubMed1995

Dynamic localization of RNase MRP RNA in the nucleolus observed by fluorescent RNA cytochemistry in living cells.

The dynamic intra-nuclear localization of MRP RNA, the RNA component of the ribonucleoprotein enzyme RNase MRP, was examined in living cells by the method of fluorescent RNA cytochemistry (Wang, J., L.-G. Cao, Y.-L. Wang, and T. Pederson. 1991. Proc. Natl. Acad. Sci. USA. 88:7391-7395). MRP RNA very rapidly accumulated in nucleoli after nuclear microinjection of normal rat kidney (NRK) epithelial cells. Localization was specifically in the dense fibrillar component of the nucleolus, as revealed by immunocytochemistry with a monoclonal antibody against fibrillarin, a known dense fibrillar component protein, as well as by digital optical sectioning microscopy and 3-D stereo reconstruction. When MRP RNA was injected into the cytoplasm it was not imported into the nucleus. Nuclear microinjection of mutant MRP RNAs revealed that nucleolar localization requires a sequence element (nucleotides 23-62) previously implicated as a binding site for a nucleolar protein, the To antigen. These results demonstrate the dynamic localization of MRP RNA in the nucleus and provide important insights into the nucleolar targeting of MRP RNA.

Animals↗
PubMed1994

Pseudouridine formation in U2 small nuclear RNA.

U2 small nuclear RNA contains 13 pseudouridine (psi) nucleotides, of which 11 are clustered in 5' regions involved in base-pairing interactions with other RNAs in the spliceosome. As a first step toward understanding the psi formation pathway in U2 RNA, we investigated psi formation on unmodified human U2 RNA in a HeLa cell extract system. Psi formation was found to occur specifically within only those RNase T1 oligonucleotide fragments of U2 RNA known to contain psi in vivo. Using 5-fluorouridine (FUrd)-containing U2 RNAs as specific inhibitors of psi formation in non-FUrd-substituted substrate U2 RNA, we found that wild-type FUrd-containing U2 RNA as well as several FUrd-containing mutant U2 RNAs completely inhibited psi formation. In contrast, certain other mutant U2 RNAs containing FUrd displayed reduced inhibitory capacity. In these cases psi modifications occurred in specific RNase T1 fragments of the substrate U2 RNA only if the FUrd-containing competitor RNA was mutated at or near this site. Formation of psi at one site in U2 RNA appeared to be neither dependent on prior psi formation at another site or sites nor required for subsequent psi formation elsewhere in the molecule. This autonomous mode of psi formation may be driven by multiple psi synthase enzymes acting independently at different sites in U2 RNA.

Animals↗
PubMed1993

Serine/threonine phosphorylation regulates binding of C hnRNP proteins to pre-mRNA.

The C hnRNP proteins bind to nascent pre-mRNA and are thought to participate in an early step of the pre-mRNA splicing pathway. We report here that C hnRNP proteins are phosphorylated by a casein kinase II activity in a HeLa cell nuclear extract and that dephosphorylation of C hnRNP proteins is inhibited by the specific protein-serine/threonine-phosphatase 1/2A inhibitor okadaic acid. We further find that dephosphorylation of C hnRNP proteins is required for their binding to adenovirus and human beta-globin pre-mRNAs. These results indicate that the participation of C hnRNP proteins in pre-spliceosome assembly is coupled to a dynamic cycle of their phosphorylation and dephosphorylation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1993

U2 small nuclear RNA 3' end formation is directed by a critical internal structure distinct from the processing site.

Mature U2 small nuclear RNA is generated by the removal of 11 to 12 nucleotides from the 3' end of the primary transcript. This pre-U2 RNA processing reaction takes place in the cytoplasm. In this study, the sequences and/or structures of pre-U2 RNA that are important for 3' processing have been examined in an in vitro system. The 7-methylguanosine cap, stem-loops I and II, the lariat branch site recognition sequence, the conserved Sm domain, and several other regions throughout the 5' end of U2 RNA have no apparent role in the 3' processing reaction. In fact, deletion of the entire first 104 nucleotides resulted in mini-pre-U2 RNAs which were efficiently processed. Similarly, deletion of the top two-thirds of stem-loop III or mutation of nucleotides in the loop of stem-loop IV had little effect on 3' processing. Most surprisingly, the precursor's 11- to 12-nucleotide 3' extension itself was of relatively little importance, since this sequence could be replaced with completely different sequences with only a minor effect on the 3' processing reaction. In contrast, we have defined a critical structure consisting of the bottom of stem III and the stem of stem-loop IV that is essential for 3' processing of pre-U2 RNA. Compensatory mutations which restore base pairing in this region resulted in normal 3' processing. Thus, although the U2 RNA processing activity recognizes the bottom of stem III and stem IV, the sequence of this critical region is much less important than its structure. These results, together with the surprising observation that the reaction is relatively indifferent to the sequence of the 11- to 12-nucleotide 3' extension itself, point to a 3' processing reaction of pre-U2 RNA that has sequence and structure requirements significantly different from those previously identified for pre-mRNA 3' processing.

Base Sequence↗