Identification and expression-profiling of Xenopus tropicalis miRNAs including plant miRNA-like RNAs at metamorphosis

Small RNAs (miR159-like RNAs) identical to some plant miR159s were found in Xenopus tropicalis miRNA cDNA libraries (30 miRNA families consisting of 75 unique sequences). Preferential expression of this RNA species was found in neural tissues during development. A sequence matching to this RNA species was not found in the 21 available animal's genome databases, but its resembling sequences associated with transposons were found in the X. tropicalis database. A possibility of horizontal transfer of the miR159 genes from plants will be discussed. Expression profiles of other miRNA species at metamorphosis were shown by DNA array and/or Northern hybridization.     

Cloning and expression analysis of piRNA-like RNAs: adult testis-specific small RNAs in chicken

Many small RNAs have been cloned from animal gonads, for example, endogenous small interfering RNAs (endo-siRNAs) were found in oocytes and piwi-interacting RNAs (piRNAs) were found in testis. Gallus gallus (chicken) is an important model organism, but few small RNAs have been identified from its gonads. In this study, we isolated and cloned 156 small RNAs from adult chicken testes. Since there is a reasonably even distribution from 22 to 33 nt, these small RNAs are slightly longer than miRNAs and endo-siRNAs. Genome mapping indicated that these small RNAs were derived from intergenic regions, exons, introns, and repetitive elements including chicken repeat 1, long terminal repeats, and simple repeats. Since they are similar with piRNAs, we named them piRNA-like RNAs (pil-RNAs). Northern blotting of 16 selected sequences showed that nine are specifically expressed in the adult testis. The vast majority of these pil-RNAs are poorly conserved between species, suggesting that they are unique to the adult chicken testis. Further analysis of the cloned pil-RNAs will improve our understanding of the function of small RNAs in animal gonad development.     

Tumorigenic Xenopus cells express several maternal and early embryonic mRNAs

Recombinant cDNA libraries were constructed from poly(A)+ RNA isolated from different stages of oogenesis and embryogenesis from the clawed toad Xenopus laevis. Hybridization analyses were used to describe the accumulation of specific RNAs represented by these cDNA clones in oocytes, embryos, adult liver, a cell line derived from Xenopus borealis embryos (Xb693), and a tumorigenic substrain of that cell line (Xb693T). It was found that from 550 cDNA clones analysed, six sequences accumulate to higher titers in poly(A)+ RNA isolated from the tumorigenic cell line compared with the non-tumorigenic cell line. All six sequences were expressed at high levels during oogenesis, and the titers of three of these sequences decreased considerably during oogenesis. DNA sequencing of these three sequences followed by a computer search of protein data banks has identified them as coding for the glycolytic enzyme enolase, the ATP-ADP carrier protein, and a-tubulin.     

Structure and expression of the nerve growth factor gene in Xenopus oocytes and embryos

A large part of the coding portion of the Xenopus nerve growth factor (NGF) gene has been identified and cloned by the use of a chicken cDNA probe and its sequence has been determined. Comparison of the derived amino acid sequence of mature Xenopus NGF with that of other species showed a high conservation, whereas comparison of the prepropeptide showed large divergent regions alternated with short conserved regions. Expression of the NGF gene was examined during development of oocytes and embryos. Surprisingly, NGF mRNA was found in the oocyte; it is present in small previtellogenic as well as in fully grown oocytes. NGF mRNA, passed to the embryo at fertilization, is degraded before the gastrula stage and starts accumulating again around the stage of the neurula. The association of NGF mRNA with polysomes is indicative of NGF synthesis during oogenesis. In fact, by using antibodies against mouse NGF it was possible to reveal NGF molecules present as precursors. These molecules accumulate during oogenesis and are maintained in the embryos up to the blastula stage; a very faint band corresponding to a smaller size peptide is sometimes detected. A maternal role for the NGF can be proposed, although a possible activity of NGF in the oocyte cannot be ruled out.     

Small nuclear U-ribonucleoproteins in Xenopus laevis development. Uncoupled accumulation of the protein and RNA components

The accumulation of protein and RNA components of small nuclear U-ribonucleoprotein particles is non-co-ordinate during oogenesis and early embryogenesis in Xenopus laevis. Northern blot hybridization of a cloned Xenopus U2-RNA gene to oocyte and embryo RNAs demonstrates that the amount of small nuclear U2-RNA per oocyte reaches a plateau early in oogenesis (at the start of yolk deposition); further accumulation is not observed in oogenesis, nor in embryogenesis until the late blastula stage. In contrast, we show by immunoblot analysis that the proteins that bind to small nuclear U-RNAs continue to be accumulated after vitellogenesis begins, reaching maximum amounts only at the end of oocyte development. No further accumulation of these proteins is seen during embryogenesis. The consequences of this non-co-ordinate synthesis of small nuclear RNA and small nuclear RNA-binding proteins are as follows: a 10- to 20-fold excess of the protein components of the small ribonucleoprotein particles over small nuclear RNA exists in large oocytes; the bulk of the protein is cytoplasmic, while the RNA is nuclear. Thus the excess protein in the cytoplasm is uncomplexed with RNA. The imbalance between protein and RNA is not corrected until the late blastula or early gastrula stages of embryogenesis, when a tenfold increase in the amount of small nuclear U2-RNA is detected. Thus the protein, but not the RNA, components of small nuclear U-ribonucleoprotein particles are stockpiled in oocytes for later use in embryonic development. During the course of these studies, we also found that there are tissue-specific differences in the Sm-antigenic proteins of X. laevis.     

Translational control during early development

Early development in many animals is programmed by maternally inherited messenger RNAs. Many of these mRNAs are translationally dormant in immature oocytes, but are recruited onto polysomes during meiotic maturation, fertilization, or early embryogenesis. In contrast, other mRNAs that are translated in oocytes are released from polysomes during these later stages of development. Recent studies have begun to define the cis and trans elements that regulate both translational repression and translational induction of maternal mRNA. The inhibition of translation of some mRNAs during early development is controlled by discrete sequences residing in the 3' and 5' untranslated regions, respectively. The translation of other RNAs is due to polyadenylation which, at least in oocytes of the frog Xenopus laevis, is regulated by a U-rich cytoplasmic polyadenylation element (CPE). Although similar, the CPE sequences of various mRNAs are sufficiently different to be bound by different proteins. Two of these proteins and their interactions are described here.     

Posttranscriptional regulation of c-myc RNA during early development of Xenopus laevis

The remarkable stability of c-myc during oogenesis contrasts with its degradation during the early developmental period in Xenopus laevis. Three evolutionary conserved motifs found in the 3'-untranslated region of Xenopus c-myc RNAs have been analyzed for a possible role in c-myc RNA degradation. No specific degradation was observed when these sequences were cloned downstream of a reporter gene and the corresponding RNAs were injected into fertilized eggs. The relation between polyadenylation and degradation of c-myc mRNA has been examined during early development. c-myc is adenylated during early oogenesis, and a dramatic de-adenylation occurs in full grown oocytes. Consequently, the de-adenylation of c-myc mRNA that occurs in eggs might be a requirement for its degradation after fertilization, but is not sufficient to trigger its degradation.     

Nucleocytoplasmic distribution of snRNPs and stockpiled snRNA-binding proteins during oogenesis and early development in Xenopus laevis

The distribution of small nuclear ribonucleoprotein particles containing U snRNAs (U snRNPs) during oogenesis and early development in Xenopus was analyzed with a lupus antibody (anti-Sm) that reacts with snRNA-binding proteins. Fully grown oocytes and embryos prior to gastrulation were found to be relatively depleted of U snRNPs in their nuclei and to contain an excess of snRNA-binding proteins stored in the cytoplasm. During late blastula-early gastrula, or after microinjection of U snRNAs into the cytoplasm of a mature oocyte, the proteins migrate into the nucleus. Dot hybridization analysis showed that small previtellogenic oocytes already contain a maximal amount of U1 (and U2) snRNAs, which then decreases to about 20% of that value in fully mature oocytes, even though the cell's volume has increased enormously. Thus fully grown oocytes and eggs accumulate snRNA-binding proteins for use during early development, but this is not coupled with the accumulation of U snRNA.     

Identification of novel microRNAs in Xenopus laevis metaphase II arrested eggs

Using a combination of deep sequencing and bioinformatics approach, we for the first time identify miRNAs and their relative abundance in mature, metaphase II arrested eggs in Xenopus laevis. We characterize 115 miRNAs that have been described either in Xenopus tropicalis (85), X. laevis (9), or other vertebrate species (21) that also map to known Xenopus pre-miRNAs and to the X. tropicalis genome. In addition, 72 new X. laevis putative candidate miRNAs are identified based on mapping to X. tropicalis genome within regions that have the propensity to form hairpin loops. These data expand on the availability of genetic information in X. laevis and identify target miRNAs for future functional studies.     

Biochemical characterization of a cellular structure retaining vegetally localized RNAs in Xenopus late stage oocytes

Two pathways operate during Xenopus oogenesis to localize a small number of RNAs to the vegetal cortex. Correct localization of these RNAs is essential to normal development as the proteins they encode are involved in specifying cell type and in patterning the early embryo. Binding these RNAs to the vegetal cortex and thus preserving their localized condition is a critical step, although little is known about how this is achieved. In this study, we have used a biochemical approach to examine the anchoring step. Xlsirts, an abundant localized RNA (locRNA), was selectively enriched in a detergent-insoluble fraction (DIF) prepared from oocytes that had completed the RNA localization process. These putative RNA-anchoring complexes were analyzed by density gradient centrifugation and in RNA-protein binding assays. Cortical Xlsirts and other localized RNAs are specifically found in the heavy region of sucrose gradients and in the pellet, quite different from other cellular RNPs. Four proteins were identified by UV-crosslinking that bound the Xlsirts localization signal in the cortex, but not in the soluble fraction. These are likely members of the anchoring complex and appear to include vera, a characterized Vg1 RNA binding protein. Vera was found to co-sediment with other locRNAs found in the vegetal cortex, suggesting that it is a common component of locRNPs. Finally, we found that locRNPs extracted into the soluble fraction had the same buoyant density as typical ooplasmic RNPs. We propose that locRNAs are organized and anchored in the cortex as typical RNPs.     

Archaeal guide RNAs function in rRNA modification in the eukaryotic nucleus

In eukaryotes, many Box C/D small nucleolar RNAs base pair with ribosomal RNA through short complementary guide sequences, thereby marking up to 100 individual nucleotides of ribosomal RNA for 2'-O-methylation. Function of the eukaryotic Box C/D RNAs depends upon interaction with at least six known proteins. Box C/D RNAs are not known to exist in Bacteria but were recently identified in Archaea by biochemical analysis and computational genomic screens and have likely evolved independently in Archaea and Eukarya for more than 2000 million years. We have microinjected Box C/D RNAs from Pyrococcus furiosus, a hyperthermophilic archaeon, into the nuclei of oocytes from the aquatic frog Xenopus laevis. Our results show that Box C/D RNAs derived from this prokaryote are retained in the nucleus, localize to nucleoli, and interact with the X. laevis Box C/D RNA binding proteins fibrillarin, Nop56, and Nop58. Furthermore, we have demonstrated the ability of archaeal Box C/D RNAs to direct site-specific 2'-O-methylation of ribosomal RNA. Our studies have revealed the remarkable ability of archaeal Box C/D RNAs to assemble into functional RNA-protein complexes in the eukaryotic nucleus.