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A Krol

Publications and source records attributed to A Krol.

At least 91 records · Page 5Linked to original sources
PubMed1992

The proximal promoter and the start site cooperate to specify correct U1 snRNA transcription initiation by RNA polymerase II.

In this work, we attempted to gain insight into the detailed mechanism allowing correct transcription initiation of U1 snRNA genes by RNA polymerase II. Abolition of the CA motif residing at -1/+1 in the Xenopus U1 gene leads to a loss of the ability of the promoter to direct accurate initiation. A discrete site is selected only if a purine preceded by a pyrimidine is positioned at 58/57 bp downstream of the center of the PSE. The PSE alone is unable to designate a discrete initiation site. Rather, it serves to set the location of an initiation window without discriminating suitable from unsuitable initiation sites. The latter role is devoted to a PyPu sequence positioned at -1/+1. Therefore, it is the concomitant action of the PSE and an essential PyPu positioned at the proper distance from this promoter that specifies correct U1 snRNA transcription initiation by RNA polymerase II.

Animals↗
PubMed1992

An additional long-range interaction in human U1 snRNA.

We present evidence for the existence of an additional long-range interaction in vertebrate U1 snRNAs. By submitting human U1 snRNP, HeLa nuclear extracts, authentic human or X. laevis in vitro transcribed U1 snRNAs to RNase V1, a nuclease specific for double-stranded regions, cleavages occurred in the sequence psi psi ACC (positions 5-9) residing in the 5' terminal region of the RNA. The RNase V1 sensitive region is insensitive to single-stranded probes, something unexpected knowing that it was considered single-stranded in order to base-pair to pre-mRNA 5' splice site. We have identified the sequence GGUAG (positions 132-136) as the only possible 3' partner. Mutants, either abolishing or restoring the interaction between the partners, coupled to an RNase V1 assay, served to substantiate this base-pairing model. The presence of this additional helix, even detected in nuclear extracts under in vitro splicing conditions, implies that a conformational change must occur to release a free U1 snRNA 5' end.

Base Sequence↗
PubMed1992

The differential transcriptional activity of two amphibian U1 small-nuclear RNA genes correlates with structural differences in the proximal sequence element.

We previously analyzed the transcription of an axolotl U1 small-nuclear RNA (snRNA) gene (AmU1) by microinjection into Xenopus laevis oocytes. In such an assay, AmU1 showed a low template activity compared to that of an X. laevis U1 snRNA gene (XlU1B2). Swapping the proximal sequence element (PSE) with that of XlU1B2 was required for AmU1 to acquire a transcription level equal to that of XlU1B2. In the present work, we examine the functional importance of the nucleotides that are common or different in both PSEs with the aim of identifying which nucleotides within the Xenopus U1 PSE are critical for this enhancement of Ambystoma mexicanum U1 snRNA transcription. The PSE mutation analysis showed that the central, phylogenetically conserved C-58/C-57 doublet is absolutely required for U1 promoter activity. In the 3' portion of this element, a CGC to ATG change (positions -54/-52) which partially restores the XlU1B2 PSE sequence, enables the AmU1 gene to gain the same transcriptional activity as XlU1B2. Remarkably, in this clustered point mutation, the sole C-54 to A-54 change is sufficient to obtain this increased level. Therefore, the activity of the AmU1 gene in injected Xenopus oocytes is strongly affected by a single sequence difference between AmU1 and XlU1B2 PSEs. This finding underscores the crucial importance of the nucleotide identity at position -54 to the function of the Xenopus U1 PSE.

Ambystoma↗
PubMed1992

Optimal tRNA((Ser)Sec) gene activity requires an upstream SPH motif.

The X. laevis tRNA((Ser)Sec) gene is different from the other tRNA genes in that its promoter contains two external elements, a PSE and a TATA box functionally equivalent to those of the U6 snRNA gene. Of the two internal promoters governing classical tRNA gene transcription, only subsists the internal B box. In this report, we show that the tRNA((Ser)Sec) contains in addition an activator element (AE) which we have mapped by extensive mutagenesis. Activation is only dependent on a 15 bp fragment residing between -209 and -195 and containing an SPH motif. In vitro, this element forms a complex with a nuclear protein which is different from the TEF-1 transcriptional activator that binds the SV40 Sph motifs. This AE is versatile since it shows capacity of activating a variety of genes in vivo, including U1 and U6 snRNAs and HSV thymidine kinase. Unexpectedly for an snRNA-related gene, the tRNA((Ser)Sec) is deprived of octamer or octamer-like motifs. The X.laevis tRNA((Ser)Sec) gene represents the first example of a Pol III snRNA-type gene whose activation of transcription is completely octamer-independent.

Animals↗
PubMed1991

Sequence, organization and transcriptional analysis of a gene encoding a U1 snRNA from the axolotl, Ambystoma mexicanum.

AmU1, a DNA fragment containing a U1 small nuclear RNA (snRNA)-encoding gene, was isolated from the axolotl, Ambystoma mexicanum. Although this U1 snRNA, produced in axolotl oocytes, exhibits the lowest degree of sequence conservation among vertebrates, its secondary structure is maintained by a number of compensatory base changes. The proximal sequence element (PSE) is only weakly similar to that of the previously characterized Xenopus laevis PSE. Exchanging either the entire upstream regions with their X. laevis U1 (XlU1) homologues or only the PSE with the XlU1 PSE increases the transcription rate of the AmU1 gene to a level similar to that of the XlU1 gene. However, while allowing the AmU1 gene to be transcribed with high efficiency in X. laevis oocytes, the strict swapping of the 12-bp constituting the XlU1 PSE does not confer competitive ability to the AmU1 gene. We present evidence that the PSE is the major, but not the only element responsible for the low template activity of the AmU1 gene in X. laevis oocytes and our data suggest that other sequences, perhaps flanking the PSE, might also influence the binding of factor(s) participating in the assembly of the transcription complex.

Ambystoma mexicanum↗
PubMed1991

The different positioning of the proximal sequence element in the Xenopus RNA polymerase II and III snRNA promoters is a key determinant which confers RNA polymerase III specificity.

We and others have previously described the TATA motif as a major determinant for Pol III specificity of the U6 promoter. Surprisingly, however, the data documented here show that the sole introduction of a TATA sequence into a U1 Pol II snRNA gene is not sufficient to confer Pol III transcription. Rather, this promoter element can mediate optimal Pol III transcription only if the PSE, the second promoter element, is shifted 4 bp upstream of the position it occupies in Pol II snRNA genes. As a result, the PSE-TATA-start site spacing introduced into the U1 Pol II gene is identical to that of the U6 gene and is strictly required to produce properly initiated Pol III transcripts. Thus, Pol II and Pol III PSEs, although similar in sequence, are not positionally equivalent. Competitive experiments raise the possibility that vertebrate U6 genes contain other, as yet unidentified, promoter elements.

Animals↗
PubMed1991

Transcription of the Xenopus laevis selenocysteine tRNA(Ser)Sec gene: a system that combines an internal B box and upstream elements also found in U6 snRNA genes.

The transcription mode of the Xenopus tRNA(Ser)Sec gene by RNA polymerase III was deciphered by injection of mutant templates into Xenopus oocyte nuclei. tRNA(Ser)Sec represents the paradigm of a new class of RNA polymerase III genes combining tRNA and U snRNA gene regulatory elements. Its promoter is tripartite, constituted by two upstream elements, a PSE and a TATA motif that are interchangeable with those of U6 snRNA genes and an internal box B as in other tRNAs. The B box enables the transcription level dependent on the upstream promoter to be increased. Data obtained indicate that U1 snRNA (Pol II) and tRNA(Ser)Sec (Pol III) genes share at least one transcription factor, implying that the border between transcription systems is less tight than expected.

Animals↗
PubMed1990

Solution structure of human U1 snRNA. Derivation of a possible three-dimensional model.

The solution structure of human U1 snRNA was investigated by using base-specific chemical probes (dimethylsulfate, carbodiimide, diethylpyrocarbonate) and RNase V1. Chemical reagents were employed under various conditions of salt and temperature and allowed information at the Watson-Crick base-pairing positions to be obtained for 66% of the U1 snRNA bases. Double-stranded or stacked regions were examined with RNase V1. The dat gained from these experiments extend and support the previous 2D model for U1snRNA. However, to elucidate some aspects of the solution data that could not be accounted for by the secondary structure model, the information gathered from structure probing was used to provide the experimental basis required to construct and to test a tertiary structure model by computer graphics modeling. As a result, U1 snRNA is shown to adopt an asymmetrical X-shape that is formed by two helical domains, each one being generated by coaxial stacking of helices at the U1 snRNA cruciform. Chemical reactivities and model building show that a few nucleotides, previously proposed to be unpaired, can form A.G and U.U non Watson-Crick base-pairs, notably in stem-loop B. The structural model we propose for regions G12 to A124 integrates stereochemical constraints and is based both on solution structure data and sequence comparisons between U1 snRNAs.

Alkylating Agents↗
PubMed1990

Structure-probing of U1 snRNPs gradually depleted of the U1-specific proteins A, C and 70k. Evidence that A interacts differentially with developmentally regulated mouse U1 snRNA variants.

The interaction of the U1-specific proteins 70k, A and C with U1 snRNP was studied by depleting gradually U1 snRNPs of the U1-specific proteins by Mono-Q chromatography at elevated temperatures (20-37 degrees C). U1 snRNP species were obtained which were selectively depleted of either protein C, A, C and A, or of all three U1-specific proteins C, A and 70k while retaining the common proteins B' to G. These various types of U1 snRNP particles were used to study the differential accessibility of defined regions of U1 RNA towards nucleases V1 and S1 dependent on the U1 snRNP protein composition. The data indicate that in the U1 snRNP protein 70k interacts with stem/loop A and protein A with stem/loop B of U1 RNA. The presence or absence of protein C did not affect the nuclease digestion patterns of U1 RNA. Our results suggest further that the binding of protein A to the U1 snRNP particle should be independent of proteins 70k and C. Mouse cells contain two U1 RNA species, U1a and U1b, which differ in the structure of stem/loop B, with U1a exhibiting the same stem/loop B sequence as U1 RNA from HeLa cells. When we used Mono Q chromatography to investigate possible structural differences in the two types of U1 snRNPs, we observed that protein A was always preferentially lost from U1b snRNP as compared to U1a snRNPs. This indicates that one consequence of the structural difference between U1a and U1b is a lowering of the strength of binding of protein A to U1b snRNP. The possible functional significance of this finding is discussed with respect to the fact that U1b RNA is preferentially expressed in embryonal cells.

Animals↗
PubMed1990

Saccharomyces cerevisiae U1 small nuclear RNA secondary structure contains both universal and yeast-specific domains.

The five small nuclear RNAs (snRNAs) involved in mammalian pre-mRNA splicing (U1, U2, U4, U5, and U6) are well conserved in length, sequence, and especially secondary structure. These five snRNAs from Saccharomyces cerevisiae show notable size and sequence differences from their metazoan counterparts. This is most striking for the large S. cerevisiae U1 and U2 snRNAs, for which no secondary structure models currently exist. Because of the importance of U1 snRNA in the early steps of "spliceosome" assembly, we wanted to compare the highly conserved secondary structure of metazoan U1 snRNA (approximately 165 nucleotides) with that of S. cerevisiae U1 snRNA (568 nucleotides). To this end, we have cloned and sequenced the U1 gene from two other yeast species possessing large U1 RNAs. Using computer-derived structure predictions, phylogenetic comparisons, and structure probing, we have arrived at a secondary structure model for S. cerevisiae U1 snRNA. The results show that most elements of higher eukaryotic U1 snRNA secondary structure are conserved in S. cerevisiae. The hundreds of "extra" nucleotides of yeast U1 RNA, also highly structured, suggest that large insertions and/or deletions have occurred during the evolution of the U1 gene.

Base Sequence↗