Structure of the sea urchin U1 RNA repeat.

The genes coding for U1 RNA in the sea urchin L. variegatus are present in a 1400 base pair tandem repeat. One member of the repeat has been cloned and its sequence determined. The repeat unit contains a single copy of the gene for L. variegatus U1 RNA. This gene encodes an RNA which is 75% homologous to mammalian U1 RNA. The L. variegatus U1 RNA could assume a secondary structure similar to that proposed for other U1 RNAs. In addition the L. variegatus U1 RNA is precipitated by anti-SM and anti-RNP antisera. Analysis of the L. variegatus genomic DNA using the cloned U1 gene as a probe reveals a major and a minor type of repeat unit. The two repeated units are the same length but differ in a number of restriction enzyme sites clustered 200-500 bases down-stream from the gene. The monomer we have cloned and sequenced is a representative of the minor repeat. A sequence (GATAA) which is -41 to -37 bases 5' to the gene has homology to the putative RNA polymerase II promoter. Fifteen bases 3' of the gene is a sequence (CAAAGAAAGAAAA) which is very similar to the sequence found 3' of the sea urchin histone genes. The two Hha I, Hpa II and Ava I sites in the repeat are all unmethylated in sperm DNA.     

The cDNA sequences of the sea urchin U7 small nuclear RNA suggest specific contacts between histone mRNA precursor and U7 RNA during RNA processing.

3' Processing of sea urchin H3 histone pre-mRNA depends on a small nuclear RNP which contains an RNA of nominally 60 nucleotide length, referred to below as U7 RNA. The U7 RNA can be enriched by precipitation of sea urchin U-snRNPs with human systematic lupus erythematosus antiserum of the Sm serotype. We have prepared cDNA clones of U7 RNA and determined by hybridization techniques that this RNA is present in sea urchin eggs at 30-fold lower molar concentration than U1 RNA. The RNA sequences derived from an analysis of eight U7 cDNA clones show neither homologies nor complementarities to any other know U-RNAs. The 3' portion of the presumptive RNA sequence can be folded into a stem-loop structure. The 5'-terminal sequences would be largely unstructured as free RNA. Their most striking feature is their base complementarity to the 3' conserved sequences of histone pre-mRNAs. Six out of nine bases of the conserved CAAGAAAGA sequence of the histone mRNA precursor and 13 out of 16 nucleotides from the conserved palindrome can be base paired with presumptive U7 RNA sequence, suggesting a unique hybrid structure for a processing intermediate formed from histone precursor and U7 RNA.     

The U1 snRNA gene repeat from the sea urchin (Strongylocentrotus purpuratus): the 70 kilobase tandem repeat ends directly 3'' to a U1 gene.

Lambda phage clones containing multiple copies of the 1.1 kb tandemly repeated unit of the sea urchin (S. purpuratus) U1 RNA genes were isolated from a gene library. The 1.1 kb repeat unit encodes a single copy of the predominant U1 RNA expressed in oocytes and embryos prior to the blastula stage. The tandem repeat unit is about 80 kb in size and is probably present one time per haploid genome as judged by pulsed-field electrophoresis of sperm DNA digested with restriction enzymes which do not cut in the repeat unit. Two of the phage contained DNA flanking the repeat unit as well as several repeat units. The tandem repeat unit ends just 3' to the U1 coding region. There is only limited homology in the 5' flanking region with U1 snRNA genes from the sea urchin L. variegatus.     

Structure of an unusual sea urchin U1 RNA gene cluster

Genomic clones containing multiple copies of the Lytechinus variegatus U1 gene have been isolated from a gene library in the phage lambda EMBL3. These clones contain both types of U1 RNA gene repeats interspersed in the same 15-kb fragment. In addition, about 1/3 of the repeat units contain a 260-bp insert 460 bp prior to the first nucleotide of the U1 RNA sequence. The inserted sequence is abundant in the sea urchin genome as judged by Southern blots of genomic DNA. There are no repeated sequences flanking the insert. The insert occurs at the same position in the highly conserved 5'-flanking region at which a deletion has previously been reported.     

A factor in sea urchin eggs inhibits transcription in isolated nuclei by sea urchin RNA polymerase III

Isolated nuclei from sea urchin embryos synthesize RNA at a rate comparable to other animal cell nuclei. All three RNA polymerases are active as judged by alpha-amanitin sensitivity and hybridization to specific cloned DNAs. Extracts were prepared from sea urchin eggs and embryos by extraction with 0.35 M KCl. None of the crude extracts had a large effect on total RNA synthesis. However, extracts from sea urchin eggs inhibited RNA polymerase III activity in nuclei from blastula and gastrula embryos. There was no effect on the synthesis of ribosomal RNA by RNA polymerase I or on the synthesis of two RNA polymerase II products, histone mRNA and the sea urchin analogue of U1 RNA. The inhibitor is present in two different species of sea urchin and has been 50-fold purified by diethylaminoethylcellulose and hydroxylapatite chromatography. The inhibitor is not present in extracts prepared from sea urchin blastula embryos.     

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.     

Functional analysis of the sea urchin U7 small nuclear RNA.

U7 small nuclear RNA (snRNA) is an essential component of the RNA-processing machinery which generates the 3' end of mature histone mRNA in the sea urchin. The U7 small nuclear ribonucleoprotein particle (snRNP) is classified as a member of the Sm-type U snRNP family by virtue of its recognition by both anti-trimethylguanosine and anti-Sm antibodies. We analyzed the function-structure relationship of the U7 snRNP by mutagenesis experiments. These suggested that the U7 snRNP of the sea urchin is composed of three important domains. The first domain encompasses the 5'-terminal sequences, up to about nucleotides 7, which are accessible to micrococcal nuclease, while the remainder of the RNA is highly protected and hence presumably bound by proteins. This region contains the sequence complementarities between the U7 snRNA and the histone pre-mRNA which have previously been shown to be required for 3' processing (F. Schaufele, G. M. Gilmartin, W. Bannwarth, and M. L. Birnstiel, Nature [London] 323:777-781, 1986). Nucleotides 9 to 20 constitute a second domain which includes sequences for Sm protein binding. The complementarities between the U7 snRNA sequences in this region and the terminal palindrome of the histone mRNA appear to be fortuitous and play only a secondary, if any, role in 3' processing. The third domain is composed of the terminal palindrome of U7 snRNA, the secondary structure of which must be maintained for the U7 snRNP to function, but its sequence can be drastically altered without any observable effect on snRNP assembly or 3' processing.     

Structural and functional characterization of mouse U7 small nuclear RNA active in 3'' processing of histone pre-mRNA.

Oligonucleotides derived from the spacer element of the histone RNA 3' processing signal were used to characterize mouse U7 small nuclear RNA (snRNA), i.e., the snRNA component active in 3' processing of histone pre-mRNA. Under RNase H conditions, such oligonucleotides inhibited the processing reaction, indicating the formation of a DNA-RNA hybrid with a functional ribonucleoprotein component. Moreover, these oligonucleotides hybridized to a single nuclear RNA species of approximately 65 nucleotides. The sequence of this RNA was determined by primer extension experiments and was found to bear several structural similarities with sea urchin U7 snRNA. The comparison of mouse and sea urchin U7 snRNA structures yields some further insight into the mechanism of histone RNA 3' processing.     

Specific contacts between mammalian U7 snRNA and histone precursor RNA are indispensable for the in vitro 3'' RNA processing reaction.

We have made a detailed molecular analysis of the reactions leading to the formation of mature 3' ends in mammalian histone mRNAs. Using two analytical protocols we have identified an essential sequence motif in the downstream spacer which is consistently present, albeit in diffuse form, mammalian histone genes. Tampering with this sequence element completely abolishes 3' processing. However, 3' cleavage in vitro, although at a very much reduced rate, can be detected when the conserved hairpin is deleted from histone precursor mRNAs. U7 snRNA, previously shown to be essential for the maturation of sea urchin histone messages, was isolated from murine cells and the sequence was determined. The approximately 63-nucleotide, trimethyl-G-capped, murine U7 snRNA possesses a sequence shown in the sea urchin U7 to be required for Sm-precipitability, and like the sea urchin U7, the 3' end of murine U7 is encased in a hairpin structure. The 5' sequence of murine U7 exhibits extensive sequence complementarity to the conserved downstream motif of the histone precursor. As expected, oligo-nucleotide-directed RNase H cleavage of this portion of murine U7 inhibits the in vitro processing reaction. These experiments identify a set of specific contacts between mammalian U7 and histone precursor RNA which is indispensable for the maturation reaction.     

A developmental switch in sea urchin U1 RNA

The sequence of U1 RNA has been determined in the eggs and embryos of two sea urchins, Lytechinus variegatus and Strongylocentrotus purpuratus. In both species the sequence of the U1 RNA changes as the embryos progress through development. The sequence of the major U1 RNA in the eggs of the two species differs in two nucleotides, while the sequence of the U1 RNA present in the late embryos and somatic tissue is identical in the two species. The U1 RNA in eggs and early embryos is primarily derived from the tandemly repeated gene set, which is not expressed in somatic tissues.     

A conserved region in the sea urchin U1 snRNA promoter interacts with a developmentally regulated factor.

The expression of the sea urchin L. variegatus U1 snRNA gene is temporally regulated during embryogenesis. Using a microinjection assay we show that a region between 203 and 345 nts 5' of the gene is required for expression. There are four conserved regions between two sea urchin species in the 345 nts 5' to the U1 gene. One region, located at about -300, binds a protein factor which is present in blastula but not gastrula nuclei. Three other potential protein binding sites within the first 200 nts 5' to the gene have been identified using a mobility shift assay and/or DNase I footprinting. Two of these regions bind factors which are not developmentally regulated and one binds a factor which is developmentally regulated. It is likely that the factor which binds at -300 is involved in expression and developmental regulation of the sea urchin U1 snRNA gene.     

Isolation and characterization of developmentally regulated sea urchin U2 snRNA genes

Genes encoding the U2 snRNA have been isolated from the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. Representatives of tandemly repeated gene sets have been isolated from both sea urchin species and a unique U2 gene has also been isolated from L. variegatus. The sequence of the U2 snRNA encoded by the tandemly repeated genes differs in two nucleotides between S. purpuratus and L. variegatus. The unique U2 gene from L. variegatus encodes the same U2 RNA as the tandemly repeated genes. There is a change in the U2 genes expressed between morula and pluteus embryos as judged by a change in the U2 RNA sequence in S. purpuratus embryos. The tandemly repeated genes were expressed at a higher rate in blastula than in gastrula stage relative to the single-copy gene, when the two genes were injected into sea urchin zygotes.