Tau exon 2 responsive elements deregulated in myotonic dystrophy type I are proximal to exon 2 and synergistically regulated by MBNL1 and MBNL2.

The splicing of the microtubule-associated protein Tau is regulated during development and is found to be deregulated in a growing number of pathological conditions such as myotonic dystrophy type I (DM1), in which a reduced number of isoforms is expressed in the adult brain. DM1 is caused by a dynamic and unstable CTG repeat expansion in the DMPK gene, resulting in an RNA bearing long CUG repeats (n>50) that accumulates in nuclear foci and sequesters CUG-binding splicing factors of the muscle blind-like (MBNL) family, involved in the splicing of Tau pre-mRNA among others. However, the precise mechanism leading to Tau mis-splicing and the role of MBNL splicing factors in this process are poorly understood. We therefore used new Tau minigenes that we developed for this purpose to determine how MBNL1 and MBNL2 interact to regulate Tau exon 2 splicing. We demonstrate that an intronic region 250 nucleotides downstream of Tau exon 2 contains cis-regulatory splicing enhancers that are sensitive to MBNL and that bind directly to MBNL1. Both MBNL1 and MBNL2 act as enhancers of Tau exon 2 inclusion. Intriguingly, the interaction of MBNL1 and MBNL2 is required to fully reverse the mis-splicing of Tau exon 2 induced by the trans-dominant effect of long CUG repeats, similar to the DM1 condition. In conclusion, both MBNL1 and MBNL2 are involved in the regulation of Tau exon 2 splicing and the mis-splicing of Tau in DM1 is due to the combined inactivation of both.

Analysis of sequence and structural features that identify the B/C motif of U3 small nucleolar RNA as the recognition site for the Snu13p-Rrp9p protein pair.

The eukaryal Snu13p/15.5K protein binds K-turn motifs in U4 snRNA and snoRNAs. Two Snu13p/15.5K molecules bind the nucleolar U3 snoRNA required for the early steps of preribosomal processing. Binding of one molecule on the C'/D motif allows association of proteins Nop1p, Nop56p, and Nop58p, whereas binding of the second molecule on the B/C motif allows Rrp9p recruitment. To understand how the Snu13p-Rrp9p pair recognizes the B/C motif, we first improved the identification of RNA determinants required for Snu13p binding by experiments using the systematic evolution of ligands by exponential enrichment. This demonstrated the importance of a U.U pair stacked on the sheared pairs and revealed a direct link between Snu13p affinity and the stability of helices I and II. Sequence and structure requirements for efficient association of Rrp9p on the B/C motif were studied in yeast cells by expression of variant U3 snoRNAs and immunoselection assays. A G-C pair in stem II, a G residue at position 1 in the bulge, and a short stem I were found to be required. The data identify the in vivo function of most of the conserved residues of the U3 snoRNA B/C motif. They bring important information to understand how different K-turn motifs can recruit different sets of proteins after Snu13p association.

An intron in the genes for U3 small nucleolar RNAs of the yeast Saccharomyces cerevisiae.

The origin of the intervening sequences (introns), which are removed during RNA maturation, is currently unknown. They are found in most genes encoding messenger RNAs, but are lacking in almost all small nuclear (sn)RNAs. One exceptional snRNA (U6) is part of the spliceosomal machinery that is involved in messenger RNA maturation. It has been suggested that its intron arose as a result of incorrect splicing of a messenger RNA precursor. This study revealed the presence of an intron, with the characteristic features of nuclear introns from precursors to messenger RNA, in the two genes coding for Saccharomyces cerevisiae U3 snRNA. The branch point was GACTAAC instead of the TACTAAC sequence found in all yeast introns examined so far. As U3 is a nucleolar snRNA required for maturation of ribosomal RNA, its intron could not have been acquired from aberrant messenger RNA processing in a spliceosome.

An improved definition of the RNA-binding specificity of SECIS-binding protein 2, an essential component of the selenocysteine incorporation machinery.

By binding to SECIS elements located in the 3'-UTR of selenoprotein mRNAs, the protein SBP2 plays a key role in the assembly of the selenocysteine incorporation machinery. SBP2 contains an L7Ae/L30 RNA-binding domain similar to that of protein 15.5K/Snu13p, which binds K-turn motifs with a 3-nt bulge loop closed by a tandem of G.A and A.G pairs. Here, by SELEX experiments, we demonstrate the capacity of SBP2 to bind such K-turn motifs with a protruding U residue. However, we show that conversion of the bulge loop into an internal loop reinforces SBP2 affinity and to a greater extent RNP stability. Opposite variations were found for Snu13p. Accordingly, footprinting assays revealed strong contacts of SBP2 with helices I and II and the 5'-strand of the internal loop, as opposed to the loose interaction of Snu13p. Our data also identifies new determinants for SBP2 binding which are located in helix II. Among the L7Ae/L30 family members, these determinants are unique to SBP2. Finally, in accordance with functional data on SECIS elements, the identity of residues at positions 2 and 3 in the loop influences SBP2 affinity. Altogether, the data provide a very precise definition of the SBP2 RNA specificity.

Pseudouridine mapping in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (snRNAs) reveals that pseudouridine synthase pus1p exhibits a dual substrate specificity for U2 snRNA and tRNA.

Pseudouridine (Psi) residues were localized in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (UsnRNAs) by using the chemical mapping method. In contrast to vertebrate UsnRNAs, S. cerevisiae UsnRNAs contain only a few Psi residues, which are located in segments involved in intermolecular RNA-RNA or RNA-protein interactions. At these positions, UsnRNAs are universally modified. When yeast mutants disrupted for one of the several pseudouridine synthase genes (PUS1, PUS2, PUS3, and PUS4) or depleted in rRNA-pseudouridine synthase Cbf5p were tested for UsnRNA Psi content, only the loss of the Pus1p activity was found to affect Psi formation in spliceosomal UsnRNAs. Indeed, Psi44 formation in U2 snRNA was abolished. By using purified Pus1p enzyme and in vitro-produced U2 snRNA, Pus1p is shown here to catalyze Psi44 formation in the S. cerevisiae U2 snRNA. Thus, Pus1p is the first UsnRNA pseudouridine synthase characterized so far which exhibits a dual substrate specificity, acting on both tRNAs and U2 snRNA. As depletion of rRNA-pseudouridine synthase Cbf5p had no effect on UsnRNA Psi content, formation of Psi residues in S. cerevisiae UsnRNAs is not dependent on the Cbf5p-snoRNA guided mechanism.

Conserved loop I of U5 small nuclear RNA is dispensable for both catalytic steps of pre-mRNA splicing in HeLa nuclear extracts.

The function of conserved regions of the metazoan U5 snRNA was investigated by reconstituting U5 small nuclear ribonucleoprotein particles (snRNPs) from purified snRNP proteins and HeLa or Xenopus U5 snRNA mutants and testing their ability to restore splicing to U5-depleted nuclear extracts. Substitution of conserved nucleotides comprising internal loop 2 or deletion of internal loop 1 had no significant effect on the ability of reconstituted U5 snRNPs to complement splicing. However, deletion of internal loop 2 abolished U5 activity in splicing and spliceosome formation. Surprisingly, substitution of the invariant loop 1 nucleotides with a GAGA tetraloop had no effect on U5 activity. Furthermore, U5 snRNPs reconstituted from an RNA formed by annealing the 5' and 3' halves of the U5 snRNA, which lacked all loop 1 nucleotides, complemented both steps of splicing. Thus, in contrast to yeast, loop 1 of the human U5 snRNA is dispensable for both steps of splicing in HeLa nuclear extracts. This suggests that its function can be compensated for in vitro by other spliceosomal components: for example, by proteins associated with the U5 snRNP. Consistent with this idea, immunoprecipitation studies indicated that several functionally important U5 proteins associate stably with U5 snRNPs containing a GAGA loop 1 substitution.

Identification of the Saccharomyces cerevisiae RNA:pseudouridine synthase responsible for formation of psi(2819) in 21S mitochondrial ribosomal RNA.

So far, four RNA:pseudouridine (Psi)-synthases have been identified in yeast Saccharomyces cerevisiae. Together, they act on cytoplasmic and mitochondrial tRNAs, U2 snRNA and rRNAs from cytoplasmic ribosomes. However, RNA:Psi-synthases responsible for several U-->Psi conversions in tRNAs and UsnRNAs remained to be identified. Based on conserved amino-acid motifs in already characterised RNA:Psi-synthases, four additional open reading frames (ORFs) encoding putative RNA:Psi-synthases were identified in S.cerevisiae. Upon disruption of one of them, the YLR165c ORF, we found that the unique Psi residue normally present in the fully matured mitochondrial rRNAs (Psi(2819)in 21S rRNA) was missing, while Psi residues at all the tested pseudo-uridylation sites in cytoplasmic and mitochondrial tRNAs and in nuclear UsnRNAs were retained. The selective U-->Psi conversion at position 2819 in mitochondrial 21S rRNA was restored when the deleted yeast strain was transformed by a plasmid expressing the wild-type YLR165c ORF. Complementation was lost after point mutation (D71-->A) in the postulated active site of the YLR165c-encoded protein, indicating the direct role of the YLR165c protein in Psi(2819)synthesis in mitochondrial 21S rRNA. Hence, for nomenclature homogeneity the YLR165c ORF was renamed PUS5 and the corresponding RNA:Psi-synthase Pus5p. As already noticed for other mitochondrial RNA modification enzymes, no canonical mitochondrial targeting signal was identified in Pus5p. Our results also show that Psi(2819)in mitochondrial 21S rRNA is not essential for cell viability.

Conserved stem-loop structures in the HIV-1 RNA region containing the A3 3' splice site and its cis-regulatory element: possible involvement in RNA splicing.

The HIV-1 transcript is alternatively spliced to over 30 different mRNAs. Whether RNA secondary structure can influence HIV-1 RNA alternative splicing has not previously been examined. Here we have determined the secondary structure of the HIV-1/BRU RNA segment, containing the alternative A3, A4a, A4b, A4c and A5 3' splice sites. Site A3, required for tat mRNA production, is contained in the terminal loop of a stem-loop structure (SLS2), which is highly conserved in HIV-1 and related SIVcpz strains. The exon splicing silencer (ESS2) acting on site A3 is located in a long irregular stem-loop structure (SLS3). Two SLS3 domains were protected by nuclear components under splicing condition assays. One contains the A4c branch points and a putative SR protein binding site. The other one is adjacent to ESS2. Unexpectedly, only the 3' A residue of ESS2 was protected. The suboptimal A3 polypyrimidine tract (PPT) is base paired. Using site-directed mutagenesis and transfection of a mini-HIV-1 cDNA into HeLa cells, we found that, in a wild-type PPT context, a mutation of the A3 downstream sequence that reinforced SLS2 stability decreased site A3 utilization. This was not the case with an optimized PPT. Hence, sequence and secondary structure of the PPT may cooperate in limiting site A3 utilization.

Molecular characterization at the RNA and gene levels of U3 snoRNA from a unicellular green alga, Chlamydomonas reinhardtii.

A U3 snoRNA gene isolated from a Chlamydomonas reinhardtii (CRE:) genomic library contains putative pol III-specific transcription signals similar to those of RNA polymerase III-specific small nuclear (sn)RNA genes of higher plants. The 222 nt long CRE: U3 snoRNA was immunoprecipitated by anti-gamma-mpppN antisera, but not by anti-m(2,2,7)G antibodies, supporting the notion that it is a RNA polymerase III transcript. Tagged CRE: U3 snoRNA gene constructs were expressed in CRE: cells. Results of chemical and enzymatic structure probing of CRE: U3 snoRNA in solution and of DMS modification of CRE: U3 snoRNA under in vivo conditions revealed that the two-hairpin structure of the 5'-domain that is found in solution is no longer detected under in vivo conditions. The observed differences can be explained by the formation of several base pair interactions with the 18S and 5'-ETS parts of the pre-rRNA. A model that involves five intermolecular helices is proposed.

The in vitro and in vivo inactivation of ribosomes by virginiamycin M does not entail an alteration of 5, 16 and 23 S ribosomal RNA.

The M component of virginiamycin blocks protein synthesis by inactivating catalytically the 50 S ribosomal subunits: the in vitro interaction of 50 S with virginiamycin M, followed by removal of the antibiotic, results in a lasting damage of the particle. This enzyme-like inactivation of 50 S subunits resembles that of 30 S subunits by colicin E3, which entails the cleavage of 16 S rRNA. To explore this possibility, rRNA obtained from particles incubated in vivo and in vitro with virginiamycin M were analyzed. Electrophoretic analysis of 5, 16 and 23 S rRNA did not reveal major changes, nor did it show the appearance of additional fragments. To exclude the possibility of terminal alterations, the 5'- and 3'-extremities of these RNA were also sequenced and found unchanged. Conclusions drawn in the present work parallel those of an accompanying paper (Moureau, P., Di Giambattista, M. and Cocito, C. (1983) Biochim. Biophys. Acta 739, 164-172) describing the dissociation and reassociation of ribosomes incubated with virginiamycin M: the lasting ribosome damage by this antibiotic appears to be due to a conformational rather than to a structural alteration.

An unusual chemical reactivity of Sm site adenosines strongly correlates with proper assembly of core U snRNP particles.

The small nuclear ribonucleoprotein particles (snRNP) U1, U2, U4, and U5 contain a common set of eight Sm proteins that bind to the conserved single-stranded 5'-PuAU3-6GPu-3' (Sm binding site) region of their constituent U snRNA (small nuclear RNA), forming the Sm core RNP. Using native and in vitro reconstituted U1 snRNPs, accessibility of the RNA within the Sm core RNP to chemical structure probes was analyzed. Hydroxyl radical footprinting of in vitro reconstituted U1 snRNP demonstrated that riboses within a large continuous RNA region, including the Sm binding site, were protected. This protection was dependent on the binding of the Sm proteins. In contrast with the riboses, the phosphate groups within the Sm core site were accessible to modifying reagents. The invariant adenosine residue at the 5' end, as well as an adenosine two nucleotides downstream of the Sm binding site, showed an unexpected reactivity with dimethyl sulfate. This novel reactivity could be attributed to N7-methylation of the adenosine and was not observed in naked RNA, indicating that it is an intrinsic property of the RNA- protein interactions within the Sm core RNP. Further, this reactivity was observed concomitantly with formation of the Sm subcore intermediate during Sm core RNP assembly. As the Sm subcore can be viewed as the commitment complex in this assembly pathway, these results suggest that the peculiar reactivity of the Sm site adenosine bases may be diagnostic for proper assembly of the Sm core RNP. Consistent with this idea, a strong correlation was found between the unusual N7-A methylation sensitivity of the Sm core RNP and its ability to be imported into the nucleus of Xenopus laevis oocytes.

The small nuclear RNAs of Drosophila.

We have investigated the sequences of the major small nuclear RNAs of Drosophila cultured cells, with the objective of elucidating phylogenetically conserved primary and secondary structures by comparison of the data with previously determined sequences of these RNAs in vertebrate species. Our results reveal striking degrees of conservation between each Drosophila RNA and its vertebrate cognate, and also demonstrate blocks of homology among the Drosophila small nuclear RNAs, as previously described for vertebrates. The most conserved features include the 5' terminal region of U1 RNA, though to function in pre-mRNA splicing, most of the regions of U4 RNA recently implicated in 3' processing of pre-mRNA, and the major snRNP protein binding site ("domain A") that is also shared by vertebrate U1, U2, U4 and U5 RNAs. Several other conserved features have been revealed, suggesting additional regions of functional significance in these RNAs and also providing further insights into the evolutionary history of the small nuclear RNAs.

An in vivo and in vitro structure-function analysis of the Saccharomyces cerevisiae U3A snoRNP: protein-RNA contacts and base-pair interaction with the pre-ribosomal RNA.

The structure and accessibility of the S. cerevisiae U3A snoRNA was studied in semi-purified U3A snoRNPs using both chemical and enzymatic probes and in vivo using DMS as the probe. The results obtained show that S. cerevisiae U3A snoRNA is composed of a short 5' domain with two stem-loop structures containing the phylogenetically conserved boxes A' and A and a large cruciform 3' domain containing boxes B, C, C' and D. A precise identification of RNA-protein contacts is provided. Protection by proteins in the snoRNP and in vivo are nearly identical and were exclusively found in the 3' domain. There are two distinct protein anchoring sites: (i), box C' and its surrounding region, this site probably includes box D, (ii) the boxes B and C pair and the bases of stem-loop 2 and 4. Box C' is wrapped by the proteins. RNA-protein interactions are more loose at the level of boxes C and D and a box C and D interaction is preserved in the snoRNP. In accord with this location of the protein binding sites, an in vivo mutational analysis showed that box C' is important for U3A snoRNA accumulation, whereas mutations in the 5' domain have little effect on RNA stability. Our in vivo probing experiments strongly suggest that, in exponentially growing cells, most of the U3A snoRNA molecules are involved in the 10-bp interaction with the 5'-ETS region and in two of the interactions recently proposed with 18S rRNA sequences. Our experimental study leads to a slightly revised version of the model of interaction proposed by J. Hughes. Single-stranded segments linking the heterologous helices are highly sensitive to DMS in vivo and their functional importance was tested by a mutational analysis.

The archaeal sRNA binding protein L7Ae has a 3D structure very similar to that of its eukaryal counterpart while having a broader RNA-binding specificity.

The ribosomal L7Ae protein of archaea has the peculiarity to be a component of the C/D and H/ACA snRNPs, that guide rRNA post-transcriptional modifications. Its yeast (Snu13p) and human (15.5kDa protein) homologs are only found in C/D snoRNPs and the (U4/U6, U5) spliceosomal tri-snRNP. By using a large variety of RNAs, we compared the RNA-binding specificities of the recombinant Pyrococcus abyssi L7Ae and Saccharomyces cerevisiae Snu13 proteins. Unlike Snu13p, protein L7Ae binds terminal loops closed by two A:G and G:A pairs and canonical K-turn structures with similar efficiencies, provided that the terminal loop contains at least 5nt. In contrast to Snu13p, binding of protein L7Ae to canonical K-turn structures is not dependent on the identity of the residue at position 2 in the bulge. The peculiar KT-15 motif of P. abyssi 23S rRNA, that is recognized by L7Ae, does not associate with Snu13p. To get more information on the P. abyssi L7Ae protein, we solved its X-ray structure at 1.9A resolution. In spite of their sequence divergence, the free P. abyssi and bound H. marismortui proteins were found to have highly similar structures. Only a limited number of side-chain conformational changes occur at the protein-RNA interface upon RNA binding. In particular, one ion pair that is formed by residues Glu43 and Lys46 in the free protein is disrupted in the ribosomal 50S subunit, so that, residue Glu43 can interact with the RNA residue G264. The Glu43-Lys46 ion pair of protein L7Ae belongs to a complex network of ion pairs that may participate to protein thermostability.

Circular permutation within the coenzyme binding domain of the tetrameric glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus.

A circularly permuted (cp) variant of the phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus has been constructed with N- and C-termini created within the coenzyme binding domain. The cp variant has a kcat value equal to 40% of the wild-type value, whereas Km and KD values for NAD show a threefold decrease compared to wild type. These results indicate that the folding process and the conformational changes that accompany NAD binding during the catalytic event occur efficiently in the permuted variant and that NAD binding is tighter. Reversible denaturation experiments show that the stability of the variant is only reduced by 0.7 kcal/mol compared to the wild-type enzyme. These experiments confirm and extend results obtained recently on other permuted proteins. For multimeric proteins, such as GAPDH, which harbor subunits with two structural domains, the natural location of the N- and C-termini is not a prerequisite for optimal folding and biological activity.

Nucleotide sequence determination of the DNA region coding for Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase and of the flanking DNA regions required for its expression in Escherichia coli.

The complete nucleotide sequence of a 3541-base pairs (bp) DNA fragment from Bacillus stearothermophilus able to complement an Escherichia coli glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) mutant (gapD-) has been determined. The B. stearothermophilus gap gene consists of a 1005-bp open reading frame commencing with an ATG start codon and ending with a TAA stop codon. Upstream from the start codon is a strong Shine-Dalgarno sequence typical of Gram-positive bacteria. Only one putative RNA polymerase recognition signal (-35 and -10 regions) is found 1153 bp upstream from the ATG start codon. In vivo utilization of this signal is in agreement with the study of gene expression from different subclones of the original fragment. 57 bp downstream from the TAA stop codon is a structure resembling Rho-independent transcription termination signals. Although B. stearothermophilus GAPDH-coding gene is highly expressed in E. coli, it contains several rare codons for E. coli. The predicted amino acid sequence of the GAPDH enzyme presents several differences with the amino acid sequence previously determined from the protein and is in better agreement with published crystallographic data.

Molecular cloning of the glyceraldehyde-3-phosphate dehydrogenase genes of Bacillus stearothermophilus and Escherichia coli, and their expression in Escherichia coli.

Recombinant plasmids derived from pBR322, which carry gap genes coding for the D-glyceraldehyde-3-phosphate dehydrogenases (GAPDH) of Bacillus stearothermophilus and Escherichia coli, have been isolated. The selection was carried out by complementation of an E. coli gapam mutant. Two plasmids containing B. stearothermophilus and E. coli DNA inserts of 4.3 kb and 1.4 kb, respectively, were characterized. Transformation of the E. coli mutant with either of the recombinant plasmids lead to a very high expression of the GAPDH activity. GAPDH produced by the strain containing the B. stearothermophilus gap gene was characterized by immunological cross-reactivity with antiserum raised against pure B. stearothermophilus GAPDH.

The first determination of pseudouridine residues in 23S ribosomal RNA from hyperthermophilic Archaea Sulfolobus acidocaldarius.

We describe the first identification of pseudouridine (Psi) residues in ribosomal RNA (23S rRNA) of an hyperthermophilic Archaea Sulfolobus acidocaldarius. In contrast to Eucarya rRNA, only six Psi residues were detected, which is rather close to the situation in Bacteria. However, three modified positions (Psi(2479), Psi(2535) and Psi(2550)) are unique for S. acidocaldarius. Two Psi residues at positions 2060 and 2594 are universally conserved, while one other Psi (position 2066) is also common to Eucarya. Taken together the results argue against the conservation of Psi-synthases between Archaea and Bacteria and provide a basis for the search of snoRNA-like guides for Psi formation in Archaea.

A phylogenetic study of U4 snRNA reveals the existence of an evolutionarily conserved secondary structure corresponding to 'free' U4 snRNA.

The nucleotide sequence of Physarum polycephalum U4 snRNA*** was determined and compared to published U4 snRNA sequences. The primary structure of P polycephalum U4 snRNA is closer to that of plants and animals than to that of fungi. But, both fungi and P polycephalum U4 snRNAs are missing the 3' terminal hairpin and this may be a common feature of lower eucaryote U4 snRNAs. We found that the secondary structure model we previously proposed for 'free' U4 snRNA is compatible with the various U4 snRNA sequences published. The possibility to form this tetrahelix structure is preserved by several compensatory base substitutions and by compensatory nucleotide insertions and deletions. According to this finding, association between U4 and U6 snRNAs implies the disruption of 2 internal helical structures of U4 snRNA. One has a very low free energy, but the other, which represents one-half of the helical region of the 5' hairpin, requires 4 to 5 kcal to be open. The remaining part of the 5' hairpin is maintained in the U4/U6 complex and we observed the conservation, in all U4 snRNAs studied, of a U bulge residue at the limit between the helical region which has to be melted and that which is maintained. The 3' domain of U4 snRNA is less conserved in both size and primary structure than the 5' domain; its structure is also more compact in the RNA in solution. In this domain, only the Sm binding site and the presence of a bulge nucleotide in the hairpin on the 5' side of the Sm site are conserved throughout evolution.

A unique U5-->A substitution in the Physarum polycephalum U1 snRNA: evidence at the RNA and gene levels.

The 5' terminal sequence of U1 snRNA that base-pairs with the intron 5' splice site in the course of spliceosome assembly was considered to be universally conserved. A study of the P polycephalum U1 snRNA at both RNA and gene levels shows that there are exceptions to this rule: the P polycephalum U1 snRNA has a U to A substitution at position 5, that is partially compensated by a high frequency of T residue at position +4 of introns. In contrast to the yeast genome, the P polycephalum genome contains several U1 snRNA coding sequences (about 20). They either encode the U1A snRNA expressed in microplasmodia or correspond to the previously cloned U1B coding sequence. Both coding sequences show the U5A substitution. The ratio of U1A versus U1B coding sequences is of about 3. A U1A gene was cloned. The 60 nt region upstream of the coding sequence has the same sequence as in the U1B gene. The U1B gene is probably expressed at another stage of the P polycephalum life cycle.