Sea Urchin U1 Research Articles

Common factors direct transcription through the proximal sequence elements (PSEs) of the embryonic sea urchin U1, U2, and U6 genes despite minimal similarity among the PSEs.

The proximal sequence element (PSE) for the sea urchin U6 small nuclear RNA gene has been defined. The most critical nucleotides for expression, located 61 to 64 nucleotides (nt) from the transcription start site, are 4 nt, AACT, at the 5' end of the PSE. Two nucleotide mutations in this region abolish transcription of the sea urchin U6 gene in vitro. The same two nucleotide mutations greatly reduce the binding of specific factors detected by an electrophoretic mobility shift assay. There is also a conserved AC dinucleotide 57 nt from the start site of the sea urchin U1 and U2 PSEs. The sea urchin U1 and U2 PSEs were substituted for the sea urchin U6 PSE, with the conserved AC sequences aligned with those of the U6 PSE. Both of these genes were expressed at levels higher than those observed with the wild-type U6 gene. Similar complexes are formed on the U1 and U2 PSEs, and formation of the complexes is inhibited efficiently by the U6 PSE. In addition, the E-box sequence present upstream of the PSE enhances U6 transcription from both the U1 and U2 PSEs. Finally, depletion of a nuclear extract with a DNA affinity column containing the U6 PSE sequence reduces expression of the U6 genes driven by the U6, U1, or U2 PSE but does not affect expression of the 5S rRNA gene. These data support the possibility that the same factor(s) interacts with the PSE sequences of the U1, U2, and U6 small nuclear RNA genes expressed in early sea urchin embryogenesis.

Common factors direct transcription through the proximal sequence elements (PSEs) of the embryonic sea urchin U1, U2, and U6 genes despite minimal similarity among the PSEs.

Common factors direct transcription through the proximal sequence elements (PSEs) of the embryonic sea urchin U1, U2, and U6 genes despite minimal similarity among the PSEs.

Formation of the 3' end of sea urchin U1 small nuclear RNA occurs independently of the conserved 3' box and on transcripts initiated from a histone promoter.

The formation of the 3' end of vertebrate small nuclear RNAs (snRNAs) requires that transcription initiate from an snRNA promoter. There is a loosely conserved required box 5 to 20 nucleotides (nt) 3' of the gene. The sea urchin snRNA genes contain promoter elements different from those of the vertebrate snRNAs. They also contain a characteristic 3' 15-nt sequence which is conserved among different sea urchin snRNA genes. We used microinjection of sea urchin U1 snRNA genes into sea urchin zygotes to define the sequence requirements for U1 snRNA 3'-end formation. Surprisingly, the conserved 3' box is not required for efficient 3'-end formation in vivo. Deletion analysis reveals that the 6 nt immediately 3' of the U1 snRNA are involved in 3'-end formation. Substitution analysis revealed that either these 6 nt 3' of the U1 RNA or the conserved 3' box could direct 3'-end formation. Transcripts initiated from a histone H4 promoter formed U1 3' ends about 50% as efficiently as transcripts initiated from the U1 promoter, even when the U1 end was placed in tandem with a histone 3'-processing signal, suggesting that transcription from an snRNA promoter is not necessary for formation of the 3' end of sea urchin U1 snRNA.

Formation of the 3' end of sea urchin U1 small nuclear RNA occurs independently of the conserved 3' box and on transcripts initiated from a histone promoter.

The formation of the 3' end of vertebrate small nuclear RNAs (snRNAs) requires that transcription initiate from an snRNA promoter. There is a loosely conserved required box 5 to 20 nucleotides (nt) 3' of the gene. The sea urchin snRNA genes contain promoter elements different from those of the vertebrate snRNAs. They also contain a characteristic 3' 15-nt sequence which is conserved among different sea urchin snRNA genes. We used microinjection of sea urchin U1 snRNA genes into sea urchin zygotes to define the sequence requirements for U1 snRNA 3'-end formation. Surprisingly, the conserved 3' box is not required for efficient 3'-end formation in vivo. Deletion analysis reveals that the 6 nt immediately 3' of the U1 snRNA are involved in 3'-end formation. Substitution analysis revealed that either these 6 nt 3' of the U1 RNA or the conserved 3' box could direct 3'-end formation. Transcripts initiated from a histone H4 promoter formed U1 3' ends about 50% as efficiently as transcripts initiated from the U1 promoter, even when the U1 end was placed in tandem with a histone 3'-processing signal, suggesting that transcription from an snRNA promoter is not necessary for formation of the 3' end of sea urchin U1 snRNA.

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.

Formation of the 3' end of sea urchin U1 small nuclear RNA occurs independently of the conserved 3' box and on transcripts initiated from a histone promoter.

Formation of the 3' end of sea urchin U1 small nuclear RNA occurs independently of the conserved 3' box and on transcripts initiated from a histone promoter.

Two promoter elements are necessary and sufficient for expression of the sea urchin U1 snRNA gene.

The essential elements of the sea urchin L. variegatus U1 snRNA promoter were mapped by microinjection of a U1 maxigene into sea urchin zygotes. Two elements are required for expression: a distal sequence element (DSE) located between -318 and -300 and a proximal sequence element (PSE) centered at -55. Removal or alteration of other sequences conserved in different sea urchin snRNA U1 genes, including deletion of all sequence between -90 and -273, did not affect the expression. Sequences around the start site were not required for expression. Deletion of nucleotides between the PSE and the start site resulted in initiation inside the U1 coding region, suggesting that the PSE determines the start site of transcription. There is no obvious similarity between the sequences required for the sea urchin U1 snRNA expression and the sequences required for the expression of other sea urchin snRNAs.

The mouse U7 small nuclear RNA gene promoter functions efficiently in Xenopus oocytes

The U7 small nuclear RNA forms part of the snRNP particle which is involved in 3'-processing of replication variant histone gene transcripts. It is one of a number of minor snRNP particles whose concentration in cells are 1130th or less than those of the major snRNPs. The mechanism(s) by which the levels of the minor snRNAs and their corresponding snRNPs are regulated is presently unknown. Likely possibilities include different rates of transcription, different processing rates, different assembly rates or different stabilities. The U7 snRNA is present at 1/30th to 160th the level of the major snRNAs in sea urchin and mammals[l] and although the sea urchin U7 genes have been cloned for some time and have typical snRNA transcription elements, no-one has yet directly compared the efficiencies of major and minor snRNA gene promoters. We have recently cloned the mouse U7 small nuclear RNA gene[2] and a U7 pseudogene[3] and have been using the Xenopus oocyte injection system to study

Sequence and expression of a mouse U7 snRNA type II pseudogene

The U7 snRNP functions in the 3' processing of histone pre-mRNAs of the replication variant class. The U7 snRNA is apparently the only RNA component of this minor snRNP particle. So far, the only U7 snRNA genes that have been isolated are a cluster of 5 from sea urchin. The function of the sea urchin U7 snRNP has been examined by microinjection experiments using Xenopus oocytes and the expression of these sea urchin U7 snRNA genes has been analysed by microinjection into sea urchin eggs. In our studies of U7 snRNA genes in mouse, we have isolated one of only 3 detectable U7 snRNA pseudogenes in the mouse genome and have determined its nucleotide sequence. Sequence comparisons and transcription analysis following microinjection of the gene into Xenopus oocytes supports our assignment of this isolate as a U7 snRNA type II pseudogene.

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.

In vivo and in vitro expression of U7 snRNA genes: cis- and trans-acting elements required for RNA polymerase II-directed transcription.

Three of five genes coding for U7 small nuclear (sn) RNA of the sea urchin Psammechinus miliaris were shown to be expressed during early embryogenesis by microinjection into sea urchin eggs followed by fertilization. Both in vivo and in nuclear extracts of blastula embryos, a minimal promoter of 80 bp of 5' flanking sequence is essential for their expression. Sequences upstream of position -80 enhance transcription in vivo, but not in vitro, approximately 5-fold. In vitro, transcription initiates at nucleotide +1 of the U7 snRNA and is directed by RNA polymerase II. Protein-DNA binding studies and site-directed mutagenesis demonstrate the presence of multiple proteins interacting with sequences between -57 and -26, which are essential for selection of the correct initiation site and for efficient in vitro transcription. Three of these factors recognize a TATA-like regulatory element between positions -53 and -45, suggesting a role for TATA-binding proteins in the initiation of sea urchin U7 snRNA transcription.

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.

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.

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.

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.

The inability of the Psammechinus miliaris H3 RNA to be processed in the Xenopus oocyte is associated with sequences distinct from those highly conserved amongst sea urchin histone RNAs.

3' processing of precursors of the H3 RNA of the sea urchin Psammechinus miliaris in Xenopus oocytes is dependent upon sea urchin U7 snRNA. Sequences necessary for this interaction are highly conserved in all sea urchin histone precursor RNAs (including the Psammechinus H3) which, in contrast, are efficiently processed in the Xenopus oocyte without the addition of the homologous U7 snRNA. We resolve this seeming paradox by demonstrating here that the inability of the sea urchin Psammechinus miliaris H3 histone RNA to be processed in the Xenopus oocyte is associated with nucleotides immediately 3' to the conserved downstream sea urchin histone sequence element. Thus, a sequence-specific element (or lack of it) is responsible for the poor recognition of the Psammechinus H3 precursor RNA by the Xenopus processing machinery.

Genetic complementation in the Xenopus oocyte: co-expression of sea urchin histone and U7 RNAs restores 3' processing of H3 pre-mRNA in the oocyte.

Efficient 3' processing of sea urchin histone H3 pre-mRNA in the Xenopus oocyte is dependent on co-injection of sea urchin U7 RNA or U7 RNP. We show that U7 RNP is a genuine member of the (Sm-type) U-snRNP family as judged by immunoprecipitation of the U7 RNA and U7 RNPs with polyclonal anti-trimethylguanosine and monoclonal anti-Sm antibodies. Sea urchin U7 RNA can be produced in the frog oocyte from RNA genes driven by a Xenopus U2 promoter. The transcripts thus synthesized have all the known characteristics of authentic U7 RNA. Our experiments further suggest that sequences near the 5' end of the U7 RNA mediate Sm-immunoprecipitability. Co-expression of sea urchin U7 RNA gene and H3 histone gene sequences in the frog oocyte complements faulty 3' maturation of histone pre-mRNA. This provides irrefutable evidence that the sea urchin U7 RNA identified earlier is indeed an essential component of the 3' processing of histone mRNAs.

Structure and expression of a second sea urchin U1 RNA gene repeat.

There are two tandemly repeated sets of U1 RNA genes in the sea urchin L. variegatus. Each of these genes is present in a 1.4 kb repeat defined by a HindIII site about 450 bases 5' to the gene. The sequences of a member of both repeating units (U1.1 and U1.2) has been determined. The repeats are nearly identical for 550 nucleotides 5' to the gene but show great divergence starting 30 nucleotides 3' to the gene, just after the CAAAGAAAGAAAA sequence thought to be required for 3' end formation. The other boundary between the conserved and non-conserved sequences is a polypyrimidine sequence (on the strand which codes for U1 RNA). Both of these repeats are expressed in blastula stage embryos, as judged by transcription of unique sequences 3' to the gene in nuclei isolated from blastula stage embryos. At least some of the two types of repeats are interspersed, since representatives of both repeat types on a single gamma phage isolated from a gene library. The sequence of the U1 RNA in L. variegatus eggs and embryos corresponds to the sequence of the U1 repeat.

Synthesis of U1 RNA in isolated nuclei from sea urchin embryos: U1 RNA is initiated at the first nucleotide of the RNA.

Nuclei from sea urchin blastula embryos synthesize a variety of small RNAs, one of which has identical mobility with sea urchin U1 RNA. This RNA is synthesized by RNA polymerase II and, in a hybridization-selection experiment, was selected by the cloned sea urchin U1 gene. The U1 RNA was initiated with ATP, but not GTP, in isolated nuclei with beta-S- and gamma-S-ribonucleotide triphosphates as substrates. The U1 RNA containing thiophosphate at the 5' end was not capped but accumulated as an uncapped transcript from which the thiophosphate could be removed with calf intestinal phosphatase.

Synthesis of U1 RNA in isolated nuclei from sea urchin embryos: U1 RNA is initiated at the first nucleotide of the RNA.

Nuclei from sea urchin blastula embryos synthesize a variety of small RNAs, one of which has identical mobility with sea urchin U1 RNA. This RNA is synthesized by RNA polymerase II and, in a hybridization-selection experiment, was selected by the cloned sea urchin U1 gene. The U1 RNA was initiated with ATP, but not GTP, in isolated nuclei with beta-S- and gamma-S-ribonucleotide triphosphates as substrates. The U1 RNA containing thiophosphate at the 5' end was not capped but accumulated as an uncapped transcript from which the thiophosphate could be removed with calf intestinal phosphatase.