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W Filipowicz

Publications and source records attributed to W Filipowicz.

At least 55 records · Page 3Linked to original sources
PubMed1993

Both Arabidopsis TATA binding protein (TBP) isoforms are functionally identical in RNA polymerase II and III transcription in plant cells: evidence for gene-specific changes in DNA binding specificity of TBP.

Promoters of pol II and pol III transcribed U-snRNA genes in plants have identical sequence elements comprised of a -30 TATA box and an upstream sequence element (USE), located four or three helical turns upstream of the TATA box in pol II and pol III genes, respectively; it is this difference in element spacing that determines the RNA polymerase specificity of the gene. We are interested in identifying factors binding to U-snRNA gene promoters and their role in selection of RNA polymerase. In this work we have investigated possible differences in the activity of the two TATA binding proteins (TBPs) encoded by two different TBP genes of Arabidopsis. Using mutant TBPs with altered DNA binding specificity, similar to those described previously in yeast, we show that two Arabidopsis TBP isoforms are equally active with both pol II and pol III U-snRNA genes and with an mRNA gene transfected into plant protoplasts. In contrast to yeast, where modified TBP permits transcription only from promoters containing the TGTAAA mutant of the consensus (TATAAA) TATA element, altered Arabidopsis TBPs also suppress other TATA box mutants. Similar results were obtained with human and yeast TBP mutants expressed in plant cells. Interestingly, in several cases suppression of different TATA box mutants by altered TBPs was gene or RNA polymerase specific suggesting that assembly of TBP into specific complexes containing different TBP-associated factors may alter DNA binding specificity of the protein.

Amino Acid Sequence↗
PubMed1993

Differential expression of U5snRNA gene variants in maize (Zea mays) protoplasts.

The small nuclear ribonucleoprotein particles U1, U2, U4/U6 and U5 participate in the removal of introns from pre-messenger RNAs in the nucleus. Three genes encoding U5snRNAs, the RNA moiety of U5snRNPs, have been isolated from maize. As in other plant UsnRNA gene families the three maize U5snRNA genes exhibit sequence variation. Two of the gene variants (MzU5.1 and MzU5.2) are clearly expressed after transfection into maize leaf protoplasts while the third gene variant (MzU5.3) is expressed at very low levels. These different levels of expression cannot be directly correlated with sequence changes in the highly conserved Upstream Sequence Element (USE) required for expression of Arabidopsis UsnRNA genes nor with differential stability of the U5snRNA transcripts. Further sequence elements may therefore have a role in regulating maize UsnRNA gene expression.

Base Sequence↗
PubMed1993

Activity of chimeric U small nuclear RNA (snRNA)/mRNA genes in transfected protoplasts of Nicotiana plumbaginifolia: U snRNA 3'-end formation and transcription initiation can occur independently in plants.

Formation of the 3' ends of RNA polymerase II (Pol II)-specific U small nuclear RNAs (U snRNAs) in vertebrate cells is dependent upon transcription initiation from the U snRNA gene promoter. Moreover, U snRNA promoters are unable to direct the synthesis of functional polyadenylated mRNAs. In this work, we have investigated whether U snRNA 3'-end formation and transcription initiation are also coupled in plants. We have first characterized the requirements for 3'-end formation of an Arabidopsis U2 snRNA expressed in transfected protoplasts of Nicotiana plumbaginifolia. We found that the 3'-end-adjacent sequence CA (N)3-10AGTNNAA, conserved in plant Pol II-specific U snRNA genes, is essential for the 3'-end formation of U2 transcripts and, similar to the vertebrate 3' box, is highly tolerant to mutation. The 3'-flanking regions of an Arabidopsis U5 and a maize U2 snRNA gene can effectively substitute for the Arabidopsis U2 3'-end formation signal, indicating that these signals are functionally equivalent among different Pol II-transcribed snRNA genes. The plant U snRNA 3'-end formation signal can be recognized irrespective of whether transcription initiation occurs at U snRNA or mRNA gene promoters, although efficiency of 3' box utilization is higher when transcription initiation occurs at the U snRNA promoter. Moreover, transcripts initiated from the U2 gene promoter can be spliced and polyadenylated. Transcription from a Pol III-specific plant U snRNA gene promoter is not compatible with polyadenylation. Finally, we reveal that initiation at a Pol II-specific plant U snRNA gene promoter can occur in the absence of the snRNA coding region and a functional snRNA 3'-end formation signal, demonstrating that these sequences play no role in determining the RNA polymerase specificity of plant U snRNA genes.

Arabidopsis↗
PubMed1992

Cap structure of U3 small nucleolar RNA in animal and plant cells is different. gamma-Monomethyl phosphate cap structure in plant RNA.

U3 small nucleolar RNA (snoRNA) is an abundant small RNA involved in the processing of pre-ribosomal RNA of eukaryotic cells. U3 snoRNA has been previously characterized from several sources, including human, rat, mouse, frog, fruit fly, dinoflagellates, slime mold, and yeast; in all these organisms, U3 snoRNA contains trimethylguanosine cap structure. In all instances where investigated, the trimethylguanosine-capped snRNAs including U3 snoRNA, are synthesized by RNA polymerase II. However, in higher plants, the U3 snoRNA is synthesized by RNA polymerase III and contains a cap structure different from trimethylguanosine (Kiss, T., and Solymosy, F. (1990) Nucleic Acids Res. 18, 1941-1949; Marshallsay, C., Kiss, T., and Filipowicz, W. (1990) Nucleic Acids Res. 18, 3451-3458; Kiss, T., Marshallsay, C., and Filipowicz, W. (1991) Cell 65, 517-526). In this study, we present evidence that cowpea and, most likely, tomato plant U3 snoRNA contains a methyl-pppA cap structure. These data show that the same U3 snoRNA contains different cap structures in different species and suggest that the kind of cap structure that an uridylic acid-rich small nuclear RNA contains is dependent on the RNA polymerase responsible for its synthesis. In vitro synthesized plant U3 snoRNA, with pppA or pppG as its 5' end, was converted to methyl-pppA/G cap structure in vitro when incubated with extracts prepared from wheat germ or HeLa cells. These data show that the capping machinery is conserved in organisms as evolutionarily distant as plants and mammals. Nucleotides 1-45 of tomato U3 snoRNA, which are capable of forming a stem-loop structure, are sufficient to direct the methyl cap formation in vitro.

Animals↗
PubMed1992

7-2/MRP RNAs in plant and mammalian cells: association with higher order structures in the nucleolus.

Mammalian MRP (for mitochondrial RNA processing) RNA, also known as 7-2 RNA, is a nuclear encoded small RNA which has been reported to function in two different cellular compartments: in the mitochondria and in the nucleus. The ribonucleoprotein particle which contains the 7-2/MRP RNA, called RNase MRP, has ribonucleolytic activity and shares some structural similarity with RNase P. It has been proposed that in mitochondria, the RNase MRP is responsible for endonucleolytic cleavage of primer RNA during DNA replication. We have characterized the gene and cDNAs encoding 7-2/MRP-like RNA in Arabidopsis and tobacco, and found that in plants this RNA is enriched in nucleoli but is undetectable in purified mitochondria isolated from tobacco leaves or cells grown in suspension. In glycerol gradients tobacco 7-2/MRP RNA cosediments with large approximately 80S structures possibly representing ribosomal precursors. Fractionation of HeLa cells has also revealed that 7-2/MRP resides in the nucleolus and that most of it is associated with complexes sedimenting at approximately 80S, similar to those containing the U3 nucleolar RNA which is known to participate in pre-rRNA processing. These results indicate that the 7-2/MRP ribonucleoparticle may be involved in ribosome biogenesis, in both plant and mammalian cells.

Animals↗
PubMed1992

Expression in transgenic tobacco of the bacterial neomycin phosphotransferase gene modified by intron insertions of various sizes.

A plant selectable marker gene consisting of cauliflower mosaic virus expression signals and the protein-coding sequence of bacterial neomycin phosphotransferase was modified by insertion of an intron sequence from a storage protein gene, phaseolin. Correct and efficient splicing of the resulting mosaic RNA was observed in transgenic tobacco plants. The insertion of various linkers or gradual increase of intron size by addition in both orientations of internal intron sequences from another plant gene (parsley, 4-coumarate ligase) had little or no effect on the precision of slicing. The gene activity measured by selectability assay in the protoplast transformation showed that only introns enlarged to 1161 bases and longer caused decreased selectability. The suitability of such mosaic marker genes for studies of RNA splicing, DNA recombination and early events after infection of plants with Agrobacterium is discussed.

Base Sequence↗
PubMed1992

Characterization of the U3 and U6 snRNA genes from wheat: U3 snRNA genes in monocot plants are transcribed by RNA polymerase III.

We have demonstrated recently that the genes encoding the U3 small nuclear RNA (snRNA) in dicot plants are transcribed by RNA polymerase III (pol III), and not RNA polymerase II (pol II) as in all other organisms studied to date. The U3 gene was the first example of a gene transcribed by different polymerases in different organisms. Based on phylogenetic arguments we proposed that a polymerase specificity change of the U3 snRNA gene promoter occurred during plant evolution. To map such an event we are examining the U3 gene polymerase specificity in other plant species. We report here the characterization of a U3 gene from wheat, a monocot plant. This gene contains the conserved promoter elements, USE and TATA, in a pol III-specific spacing seen also in a wheat U6 snRNA gene characterized in this report. Both the U3 and the U6 genes possess typical pol III termination signals but lack the cis element, responsible for 3'-end formation, found in all plant pol II-specific snRNA genes. In addition, expression of the U3 gene in transfected maize protoplasts is less sensitive to alpha-amanitin than a pol II-transcribed U2 gene. Based on these data we conclude that the wheat U3 gene is transcribed by pol III. This observation suggests that the postulated RNA polymerase specificity switch of the U3 gene took place prior to the divergence of angiosperm plants into monocots and dicots.

Amanitins↗
PubMed1992

Multiple plant RNA binding proteins identified by PCR: expression of cDNAs encoding RNA binding proteins targeted to chloroplasts in Nicotiana plumbaginifolia.

Pre-mRNA processing in eukaryotic cells requires the participation of multiple protein factors and ribonucleoprotein particles. One class of proteins involved in this process are RNA-binding proteins, which contain a domain of ca. 90 amino acids with a characteristic ribonucleoprotein consensus sequence (RNP-CS). A PCR approach that is suitable for the characterization of RNP-CS-type proteins is described. Fifteen different RNA-binding domains were amplified from Nicotiana tabacum (tobacco) using oligonucleotide primers specific for the sequences (K/R)G(F/Y)(G/A)FVX(F/Y) and (L/I/V)(F/Y)(V/I)(G/K)(N/G)L, which are conserved in known RNP-CS proteins. Using the tobacco domains as probes, cDNAs encoding two RNA-binding proteins, each containing two RNP-CS-type domains, were characterized in N. plumbaginifolia. The proteins, designated CP-RBP30 and CP-RBP31, are targeted to chloroplasts as demonstrated by expression of epitope-tagged cDNAs in transfected protoplasts, followed by indirect immunofluorescence. High levels of mRNA for each protein were found in leaves but not in roots, and expression of the CP-RBP31 mRNA was strongly regulated by light. The N. plumbaginifolia proteins described in this work are distinct from chloroplast RNA-binding proteins characterized recently in tobacco and spinach.

Amino Acid Sequence↗
PubMed1992

Characterization of the genes encoding U4 small nuclear RNAs in Arabidopsis thaliana.

Three genes encoding U4 small nuclear RNA (U4 snRNA) in the higher plant Arabidopsis thaliana have been isolated and characterized. Two of the genes, AtU4.1 and AtU4.2, contain all the transcriptional signals known to be essential for U-snRNA gene activity in dicot plants: the Upstream Sequence Element (USE), the -30 TATA box and the downstream 3' end formation sequence. The USE and TATA elements are centered approximately four helical DNA turns apart, a feature characteristic of RNA polymerase II-transcribed U-snRNA genes of plants. The genes AtU4.1 and AtU4.2 are actively transcribed in transfected plant protoplasts and in Arabidopsis plants. Expression of the third gene, AtU4.3, could not be demonstrated. Since this gene is missing the downstream signal important for RNA 3' end formation, it probably represents a pseudogene. The genes AtU4.1 and AtU4.2 encode 152-153 nt long RNAs which show 85-89% sequence similarity with broad bean and pea U4 RNAs and 60-65% similarity with mammalian U4 RNAs. Arabidopsis U4 and U6 snRNAs can be folded into the base-paired Y-shaped model supporting the importance of the U4/U6 interaction during pre-mRNA splicing in plants as well as animals.

Arabidopsis↗
PubMed1991

Alteration of the RNA polymerase specificity of U3 snRNA genes during evolution and in vitro.

We present evidence that the genes encoding U3 snRNA in plants are transcribed by RNA polymerase III (pol III) and not by RNA polymerase II (pol II) as in vertebrates or lower eukaryotes. The U3 gene is the only known example of a gene transcribed by different polymerases in different organisms. It is possible to convert the plant U3 gene into a functional pol II-transcribed gene by manipulating the spacing between the promoter elements and inserting a pol II-specific termination signal. Pol II-transcribed U3 RNA, containing the 5'-terminal cap different from that present in the wild-type counterpart, is packaged in transfected protoplasts into U3 snRNP precipitable with anti-fibrillarin antibodies. These findings provide further evidence for the common ancestry of the pol II and pol III transcription systems, and indicate that promoter diversification in some genes has occurred relatively recently.

Base Sequence↗
PubMed1991

Different effects of intron nucleotide composition and secondary structure on pre-mRNA splicing in monocot and dicot plants.

We have found previously that the sequences important for recognition of pre-mRNA introns in dicot plants differ from those in the introns of vertebrates and yeast. Neither a conserved branch point nor a polypyrimidine tract, found in yeast and vertebrate introns respectively, are required. Instead, AU-rich sequences, a characteristic feature of dicot plant introns, are essential. Here we show that splicing in protoplasts of maize, a monocot, differs significantly from splicing in a dicot, Nicotiana plumbaginifolia. As in the case of dicots, a conserved branch point and a polypyrimidine tract are not required for intron processing in maize. However, unlike in dicots, AU-rich sequences are not essential, although their presence facilitates splicing if the splice site sequences are not optimal. The lack of an absolute requirement for AU-rich stretches in monocot introns in reflected in the occurrence of GC-rich introns in monocots but not in dicots. We also show that maize protoplasts are able to process a mammalian intron and short introns containing stem--loops, neither of which are spliced in N.plumbaginifolia protoplasts. The ability of maize, but not of N.plumbaginifolia to process stem--loop-containing or GC-rich introns suggests that one of the functions of AU-rich sequences during splicing of dicot plant pre-mRNAs may be to minimize secondary structure within the intron.

Base Sequence↗
PubMed1990

RNA-polymerase specificity of transcription of Arabidopsis U snRNA genes determined by promoter element spacing.

Although most eukaryotic genes known to be transcribed by RNA polymerase III have intragenic promoter elements, some are similar to genes transcribed by RNA polymerase II in that they have upstream promoters (reviewed in refs 1-4). Transcription of the vertebrate U6 and 7SK RNA genes by RNA polymerase III depends exclusively upon upstream signals, some of which are indistinguishable from the elements essential for polymerase II-specific genes. In the plant Arabidopsis thaliana the promoter elements for the U6 and U2 small nuclear RNA genes, transcribed by RNA polymerases III and II respectively, are identical, comprising a -30 TATA box and an upstream element specific for small nuclear RNA genes. The distance between these elements differs, however. Here we report evidence that this separation is crucial in determining whether the genes are transcribed by polymerase II or III.

Base Sequence↗
PubMed1990

U6 snRNA genes of Arabidopsis are transcribed by RNA polymerase III but contain the same two upstream promoter elements as RNA polymerase II-transcribed U-snRNA genes.

Previously we have demonstrated that the U2 snRNA genes from the higher plant Arabidopsis thaliana contain two upstream elements, the USE with sequence RTCCCACATCG and a -30 'TATA' box, which are essential for transcription by RNA polymerase II, and that the conserved spacing of about four helical DNA turns between these elements is important for optimal promoter function. We have now isolated three genes encoding U6 RNA in Arabidopsis. Transcription of these genes in transfected protoplasts of Nicotiana plumbaginifolia is resistant to alpha-amanitin indicating that they are transcribed by RNA polymerase III. The upstream regions of three Arabidopsis U6 genes contain USE and -30 TATA-like elements similar to those found to be important for transcription of U2 RNA genes but the spacing between the two elements is about 10 bp closer than in the U2 genes. Using synthetic U6 genes we demonstrate that the USE and TATA elements are indispensable for their transcription, the TATA boxes of U2 and U6 genes are interchangeable, and that the intragenic A box-like sequence of U6 gene is not essential. Increasing the distance between the USE and TATA by 10 bp inactivates U6 gene transcription, demonstrating that proper positioning of the elements is also important for transcription by RNA polymerase III. The data indicate that the structure of U-snRNA gene promoters and the determinants of polymerase specificity are completely different between vertebrates and plants.

Amanitins↗
PubMed1990

Amplification of plant U3 and U6 snRNA gene sequences using primers specific for an upstream promoter element and conserved intragenic regions.

U-snRNA genes in higher plants contain two essential promoter elements, the USE with sequence RTCCCACATCG and the TATA-like box, positioned in the -70 and -30 regions, respectively. Using an oligodeoxynucleotide containing the USE motif and oligodeoxynucleotides specific for the intragenic regions conserved in U-snRNAs, several sequences encoding U6 and U3 snRNAs were determined by polymerase chain reaction (PCR) amplification of Arabidopsis thaliana and tobacco genomic DNAs. This method provides a simple and rapid procedure for characterisation of plant U-snRNA genes and their promoters. It could also be used for the characterisation of other genes containing conserved upstream promoter elements. PCR-derived fragments were used as probes for the isolation of the U3 snRNA genes from a genomic library of Arabidopsis. Two isolated U3 genes were shown to be active when transfected into protoplasts of Nicotiana plumbaginifolia. Both U3 genes contain the USE and TATA-like upstream elements located in similar positions to the U6 genes of Arabidopsis. The encoded Arabidopsis U3 snRNAs can be folded into a secondary structure which is more similar to that of U3 RNAs from lower eukaryotes rather than from metazoa.

Base Sequence↗
PubMed1990

The minimum functional length of pre-mRNA introns in monocots and dicots.

When exogenous genes are to be expressed in transgenic plants, their RNAs must be correctly processed. To gain information useful for predicting whether foreign introns will be accurately spliced, we have analysed the processing of an artificial gene in maize and Nicotiana plumbaginifolia protoplasts. A synthetic plant intron, devised to contain the elements necessary for pre-mRNA splicing in dicots, was found to be efficiently spliced in a monocot (maize) transient expression system. A series of deletion mutants of the synthetic intron was constructed to assess the minimum functional intron length. In both monocots and dicots this was found to be between 70 and 73 nt. This length requirement is similar to that seen in vertebrates, but significantly greater than that in fungi and insects.

Base Sequence↗