InteractiveFly: GeneBrief

Nucleolar protein at 60B : Biological Overview | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Nucleolar protein at 60B [Nop60B, alternatively termed Minifly (mfl)] plays a central role in ribosomal RNA processing and pseudouridylation. Eukaryotic homologs include yeast Cfb5p, rat NAP57 and human dyskerin, which encodes a gene responsible for the X-linked dyskeratosis congenita disease. Nop60B/mfl genetic analysis (Phillips, 1998; Giordano, 1999; Kauffman, 2003) represents the first in vivo functional characterization of a member of this highly conserved gene family from higher eukaryotes. In addition, Nop60B hosts an intron encoded box H/ACA snoRNA gene, the first member of this class of snoRNAs identified so far from Drosophila (Giordano, 1999). Genetic, molecular, and functional characterization of Nop60B/mfl shows that the gene is essential for Drosophila viability and fertility. While P-element induced total loss-of-function mutations cause lethality, mfl partial loss-of-function mutations cause pleiotropic defects, such as extreme reduction of body size, developmental delay, hatched abdominal cuticle, and reduced female fertility. Morphological abnormalities characteristic of apoptosis are found in the ovaries, and a proportion of eggs laid by mfl mutant females degenerates during embryogenesis. mfl has an intriguing molecular organization, hosting within its intronic sequence an intron-encoded box H/ACA snoRNA that represents the first member of this class thus far described in Drosophila. This gene has been named RNA snoH1; it may be functionally equivalent to the human U70 snoRNA (Giordano, 1999). Although null mutations in Nop60B are lethal, P element-induced alleles generate viable, but sterile flies, exhibiting severe testicular atrophy. Testicular atrophy is due to severe loss of germ cells, including stem cells, but much milder effects on the somatic cells, which are themselves maintained by a stem cell lineage. Nop60B activity is required intrinsically for the maintenance of germ-line stem cells. These phenotypes can be related to the human syndrome Dyskeratosis congenita, caused by mutations in a Nop60B homolog (Kauffman, 2003).

In eukaryotic cells, synthesis, maturation and modification of rRNA take place in the nucleolus; ribonuclearprotein particles, consisting of a variety of nucleolar proteins and small nucleolar RNAs (snoRNAs), are responsible for these essential cellular processes. Almost 100 different snoRNAs species have been identified so far in yeast and mammalian cells. Most of these snoRNAs can be classified into two major distinct families, each defined by common associated proteins and by the presence of conserved sequences, designated as either C/D or H/ACA boxes. The C and D box-containing snoRNAs display extensive sequence complementarity to conserved rRNA regions and are associated with a conserved nucleolar protein, fibrillarin or, in yeast, with the fibrillarin homolog Nop1p. Some fibrillarin-associated snoRNAs are required for rRNA processing, but most of them function as a guide in site-specific ribose methylation of rRNA (Giordano, 1999 and references therein).

Members of the other large class of snoRNAs share H and ACA elements and have only short rRNA complementary motifs, brought together by a conserved stem-loop secondary structure. This structure, composed of two hairpins connected and followed by short single-stranded regions containing the H and ACA elements, directs the site-specific pseudouridylation event with the short (5-9 nucleotide [nt]) regions of snoRNA; rRNA complementarity flanks both sides of the target site (Giordano, 1999 and references therein).

In yeast, members of the box H/ACA class of snoRNAs are specifically associated with two essential nucleolar proteins, Gar1p and Cbf5p. Gar1p, a glycine-arginine-rich protein required for accumulation of mature 18S rRNA and for rRNA pseudouridylation, is thought to play a crucial role in structuring box H/ACA sno-RNPs and favoring association of H/ACA snoRNAs to the pre-rRNA. In a two hybrid yeast assay, Gar1p interacts with Cbf5p which, in turn, coprecipitates with box H/ACA snoRNAs and is required for their stability (Lafontaine, 1998). Interestingly, Cbf5p is the yeast member of a highly conserved protein family that includes homologs from at least 18 organisms. Among eukaryotes, genetic analysis has so far been restricted to two members of this family: the yeast Cbf5 (Jiang, 1993) and the DKC1 human gene, whose mutations cause the X-linked dyskeratosis congenita disease (Heiss, 1998). Whereas little information is available on human dyskerin, Cbf5p and its rat homolog, NAP57, are known to be proteins with prevalent nucleolar localization. However, whereas it has been proposed that NAP57 may be involved in nucleo-cytoplasmatic shuttling (Meier, 1994), the yeast protein has been shown to be required for transcription, processing and efficient rRNA pseudouridylation (Cadwell, 1997; Lafontaine, 1998). This last finding raises the possibility that Cbf5p might act as eukaryotic rRNA pseudouridine synthase, a role originally suggested (Cadwell, 1997) by its homology with E. coli TruB/P35 synthase. Considering the multiple, essential functions played by Cbf5p in yeast cells, the definition of the roles played by members of this family in multicellular organisms appears to be a relevant issue that deserves extensive investigation (Giordano, 1999 and references therein).

Adding to the complexity of the function of this gene is the observation that dyskerin, the human homolog of Drosophila Nop60B, is associated not only with H/ACA small nucleolar RNAs, but also with human telomerase RNA. Sufferers of dyskeratosis congenita (DKC) have defects in highly regenerative tissues such as skin and bone marrow, chromosome instability and a predisposition to develop certain types of malignancy. Telomerase is an enzyme that adds simple sequence repeats to chromosome ends using an internal region of its associated RNA (telomerase RNA) as a template; telomerase is required for the indefinite proliferation of primary human cells. Human telomerase RNA contains an H/ACA RNA motif. Primary fibroblasts and lymphoblasts from DKC-affected males are not detectably deficient in conventional H/ACA small nucleolar RNA accumulation or function; however, DKC cells have a lower level of telomerase RNA, produce lower levels of telomerase activity and have shorter telomeres than matched normal cells. The pathology of DKC is consistent with compromised telomerase function leading to a defect in telomere maintenance, which may limit the proliferative capacity of human somatic cells in epithelia and blood (Mitchell, 1999).

The developmental time at which lethality is achieved in mfl/Nop60B mutants correlates well with MFL level. Given the similarity existing between the mfl phenotype and that caused by mutations affecting the synthesis of ribosomal components, the role of mfl on rRNA processing was examined. Electrophoresis of larval rRNA labeled by [³H]uridine incorporation shows that pre-rRNA processing is inefficient in mfl mutants. In fact, with respect to wild-type flies, increased levels of the pre-rRNA and 28S rRNA and reduced amounts of the 18S, 28Sa, and 28Sb mature species were observed. MFL over-expression in mfl transgenic flies is sufficient to reduce rRNA precursor accumulation and to increase the level of the newly synthesized 18S and 28S species (Giordano, 1999).

Northern blot analysis with three different probes derived from the rDNA internal transcribed spacer (ITS) the abnormal rRNA processing occurring in mfl/Nop60B mutants to be defined in more detail. In Drosophila the rRNA primary transcript (pre-rRNA) undergoes two alternative types of initial cleavages. The most predominant type occurs in the external transcribed spacer, at site 1, and generates the large type 'a' molecule, from which both 18S and 28S are derived. An alternative cleavage occurs within ITS, at site 3, generating the intermediate 'd' and 'b' forms which are, respectively, 18S and 28S rRNA precursors. Hybridization to a probe derived from the ITS 5' end reveals that the accumulation of the pre-rRNA observed in mfl mutants is accompanied by a reduction of the type 'a' precursor and by an increase of the 'd' form; both effects become more evident with progression of the larval development. Thus, mfl mutations specifically affect site 1 cleavage, inhibiting the formation of type 'a' molecules and the processing of the 'd' intermediate. With the pathways blocked, pre-rRNA processing proceeds mainly through pathway ß, generating equimolar amounts of 'd' and 'b' intermediate molecules. This is confirmed by hybridization to probe II, which shows that while in wild-type animals the amount of form 'b' largely exceeds that of 'd' (as expected, since the 'b' molecule is actively produced by both alpha and ß pathways), in mfl mutants these two forms are detected in similar amounts. However, since the processing of form 'd' is inhibited, this species accumulates progressively along larval development. Conversely, hybridization to probe III indicates that mfl genetic depletion does not impair site 4 cleavage of type 'b' molecule, since the amount of form c observed in the mutants exceeds even that of the control. It is concluded that form c is generated properly, but its further processing is inhibited by mfl mutations. In mfl transgenic flies, MFL over-expression leads to a reversal of all of the effects observed, although the efficiency of pre-rRNA processing is not fully restored. In heat-shocked transformed animals, in fact, MFL expression causes a decrease of pre-rRNA accumulation and an increase in the production of the type 'a' molecule. Processing of the type 'a' precursor also occurs properly, since, these larvae show an excess in form 'b' versus form d, although the amount of the 'b' molecule does not reach that observed in wild-type animals. Finally, the amount of form c appears reduced after the heat-shock, indicating that its processing is at least partially restored (Giordano, 1999).

In yeast, lack of Cbf5 gene activity affects not only rRNA processing, but also rRNA pseudouridylation. Thus, the level of modification was examined in wild-type and mfl/Nop60B mutants at several 28S and 18S pseudouridylate- (psi-) specific sites. With this aim, oligonucleotide primers complementarity was used to selected 28S or 18S regions to perform primer extension analyses on CMC-treated Drosophila rRNA, in order to localize psi residues. CMC blocks reverse transcription, resulting in a gel band terminating in one residue 3' of the Psi site. In planning these experiments, advantage was taken of the known location of Drosophila 28S rRNA pseudouridines. However, none of the 18S Psi sites examined in these experiments was previously known. In spite of the persistence of maternal rRNA, pseudouridylation appears reduced in mfl⁰⁵ larvae at several 28S sites, such as the U2442, U2444, and U2499 residues. Similar reduction was observed at various 18S rRNA sites, such as U830/U831, U840, U841, and U885, indicating that, as Cbf5, mfl is required for efficient rRNA pseudouridylation (Giordano, 1999).

An unexpected feature of the mfl/Nop60B gene structure was revealed by the finding that a small RNA species, ~0.1 kb in length, hybridized specifically with the genomic sequences of the fourth mfl intron, while it was not detected by any cDNA probe. This small RNA was detected in total RNA preparations from all developmental stages and was specifically enriched in the poly(A) minus RNA fraction. The length of the small RNA species was accurately determined on denaturing 6% polyacrylamide gels and its 5' end precisely mapped by primer extension analysis of total larval RNA using two different oligonucleotides. These experiments pointed out that this transcript was ~140 nt long and derived from position +37 to about +176 of the 235-nt-long fourth mfl intron. Since a large number of small nucleolar RNAs are intron encoded, the presence of conserved snoRNA elements within the 0.14-kb RNA sequence was examined. Two H boxes (consensus ANANNA) and a 3' terminal ACA element were found; in addition, the predicted secondary structure of the mfl intron-encoded RNA conforms well to the hairpin-hinge-hairpin-tail architecture common to most yeast and vertebrate box H/ACA snoRNAs. Two short regions of complementarity between the mfl intron encoded RNA and Drosophila 18S rRNA were also found. Short regions of pairing with rRNA are known to flank the site of pseudouridylation, allowing the positioning of the residue to be isomerized at the base of the stem, at the first unpaired position before the 3' snoRNA helical segment. The pseudouridine selected is found to be separated from the H or ACA box by 14 or, in a few cases, by 15 nucleotides. On the basis of these observations, the rRNA pairing properties of the mfl intron-encoded RNA predicted it may direct pseudouridylation of Drosophila 18S rRNA at position U1820. Primer extension analysis on CMC-treated Drosophila rRNA shows that the potentially selected residue is actually pseudouridylated. The selected U1820 residue is equivalent to U1698 of human 18S rRNA, whose pseudouridylation has been related to the U70 snoRNA. As for U1698 in human rRNA, the Drosophila U1820 residue is the first of three consecutive uridines, all of which are pseudouridylated (Giordano, 1999).

In yeast, genetic depletion of most of the box H/ACA snoRNAs has been reported to inhibit pseudouridylation of the specifically selected sites. When modification was checked of the U1820 residue in rRNA preparations obtained from mfl⁰⁵ first instar larvae, pseudouridylation was found to be reduced not only at U1820, but also at U1821 and U1822 residues. This result may be explained by the widespread inhibition of rRNA pseudouridylation observed in mfl/Nop60B mutants. Further experiments are thus required to define the specific functional role, if any, played by the mfl intron-encoded RNA (Giordano, 1999).

Finally, the localization of the mfl/Nop60B intron encoded RNA was checked by in situ hybridization experiments to whole mount ovary preparations. This analysis showed that a 0.14-kb RNA-specific antisense probe exclusively labeled the nucleoli; the signal occurs in each tested embryonic or larval tissue. Specific nucleolar localization may also be observed for MFL, whose ubiquitous expression was established using both immunolocalization and histochemical staining of lacZ activity in mfl¹ flies. In ovarian tissue preparations it was noticed that the protein occasionally diffuses into the cytoplasm in several patches of follicle cells. As judged by the presence of well defined, round-shaped nuclei having morphologically well distinguishable nucleoli, these cells should not be in or around mitosis. Moreover, cytoplasmic diffusion can also be observed after stage 10b of oogenesis, when follicular cells endocycles are reported to be terminated. It is thus plausible that occasional MFL cytoplasmic localization may be related to ability to carry out nucleolus-cytoplasmic shuttling, as proposed for NAP57 in rat cells (Giordano, 1999 and references therein).

It is concluded that mfl/Nop60B encodes an ubiquitous nucleolar protein essential for Drosophila viability and female fertility. The data also show that mfl is closely related to the other members of the Cbf5 family so far characterized from higher eukaryotes, the rat Nap57 and the human gene responsible for the X-linked dyskeratosis congenita disease. As predicted (Luzzatto, 1998), flies carrying mutations in the Drosophila DKC1 ortholog show a pleiotropic phenotype very similar to that caused by mutations that affect the synthesis of ribosomal RNA. In fact, mfl loss-of-function mutations impair rRNA processing and lead to accumulation of rRNA precursors. Although these effects are very similar to those caused by Cbf5 genetic depletion, yeast mutations preferentially affect the production of mature 18S rRNA (Lafontaine, 1998), while mfl mutations cause similar reduction of 18S and 28S rRNA species. It would be of interest to know whether this is due to a distinctive feature of Drosophila rRNA processing pathways, or whether it reflects a general property of rRNA processing in higher eukaryotes (Giordano, 1999).

In addition to affecting rRNA maturation, mfl/Nop60B loss-of-function causes reduced levels of pseudouridylation at several 28S and 18S Psi sites, suggesting that gene activity might be required for fully efficient rRNA pseudouridylation. Again, these results are reminiscent of those obtained in yeast (Lafontaine, 1998), and outline the existence of a link between rRNA processing and rRNA pseudouridylation in eukaryotes. By mapping the protein domains conserved among members of the Cbf5p family and investigating the definition of their functional roles, significant information should be generated about the functional role played by rRNA pseudouridylation, which still remains elusive. Although pseudouridylation of eukaryotic rRNAs occurs predominantly on the primary rRNA transcripts before nucleolytic processing, this type of modification is not required for efficient processing of 25S yeast rRNA. It has been suggested that pseudouridylation can contribute to rRNA folding, rRNPs assembly, and ribosomal subunit assembly. Other hypotheses, such as subtle enhancing of ribosomal functions or influencing fidelity of codon recognition, have also been proposed (Giordano, 1999 and references therein).

An additional role that could be suggested for MFL/Nop60B is based on the observation that it can occasionally diffuse within the cytoplasm. As previously suggested for NAP57 in rat cells, it is tempting to speculate that this may possibly reflect the ability of MFL to structure and export pre-ribosomal RNP particles into the cytoplasm. If confirmed, this would strongly support the view that members of this family are multifunctional proteins involved in different aspects of ribosome biogenesis. It is possible that these proteins may constitute essential components of a single multifunctional complex or, alternatively, they represent common components of structurally and functionally different RNP particles. The definition of the functional interactions required to carry out such a variety of functions will help to clarify this point (Giordano, 1999).

Remarkably, the identification and the characterization of mutations disrupting mfl gene expression has led to establishing the first animal model system for the study of the X-linked dyskeratosis congenita human disease. Some of these may immediately provide useful information for the comprehension of the molecular basis of the DKC disease. A first relevant point concerns the observation that none of the mfl mutations so far isolated disrupts the gene coding region. Thus, each Drosophila mutant line has certainly quantitative and not qualitative alterations of the gene product which causes the pleiotropic abnormalities observed. The level of MFL protein was found to be critical, and a simple dose-effect rule may be derived: when the protein level is below a crucial threshold, mortality ensues. Instead, while the protein level is lowered but still stands above a critical threshold, the viable, hypomorphic mfl¹ phenotype is reached. By analogy, it can be suggested that in humans the level of dyskerin activity may be one of the critical parameters able to trigger the DKC disease. The finding that DKC mutations mapped so far all affect the dyskerin coding region (Heiss, 1998) is in only apparent contrast with that found in Drosophila. In fact, it is reasonable to suppose that, as observed in Drosophila, total or severe loss-of-function mutations should not be compatible with life. Mutations recovered in patients might be those causing partial loss-of-function, so that the level of dyskerin activity is still compatible with survival. Accordingly, DKC patients might carry hypomorphic mutations, the human counterparts of the viable mfl¹ phenotype. Whether these hypomorphic phenotypes are simply a consequence of the inadequate mature rRNA level or are, at least partially, caused by abnormal accumulation of intermediate rRNA species is an important point which deserves further investigation. A further issue concerns the observation that, although MFL and dyskerin are ubiquitous proteins, phenotypic abnormalities are, in Drosophila as in humans, restricted to only certain tissues. Since the gene product is presumed to be critically important for protein synthesis in every cell of the body, the finding that abnormalities are developed only by selected cell types is quite surprising. However, if it is accepted that the level of protein activity may be a critical parameter, then it is reasonable to suppose that the amount of properly processed rRNA may be sufficient in cells having a slow growth rate, while in highly proliferating tissues or in cells sustaining a high rate of protein synthesis this would not the case, and degenerative cell defects could progressively be accumulated. Interestingly, inhibition of protein synthesis is known to be one of the stimuli capable of inducing apoptotic cell death, probably by decreasing the levels of essential proteins or by inhibiting the synthesis of proteins that normally suppress the spontaneous activation of apoptosis. In mfl¹ ovary, one of the Drosophila tissues where morphological abnormalities can be observed, it was found that degeneration is specifically accompanied by apoptotic cell death. This observation might also suggest a role for apoptosis in the progressive clinical manifestations of the DKC disease (Giordano, 1999).

Finally, it has been shown that mfl gene organization is intriguing; this led to the identification of the first member of the box H/ACA class of snoRNAs described so far in Drosophila. As in the case of the snoH1 gene described here, most of the snoRNAs are intron encoded, and snoRNA host genes often encode proteins involved in translation or ribosome biogenesis (reviewed by Smith, 1997). These intron-encoded snoRNAs are cotranscribed with their host pre-mRNA and their accumulation is splicing-dependent, since they are released from the excised intron by exonucleolytic processing. The observation that snoH1 RNA and mfl mRNAs levels are reduced in parallel in each mfl mutant line strongly suggests that snoH1 RNA processing is linked to the splicing of the mfl primary transcript. This feature, which allows coordinated regulation of the host protein and the intron encoded snoRNA, may hinder a precise definition of the specific functional role played by each product. With regard to snoH1, it cannot be excluded, in principle, that it may be required for Drosophila viability and that its depletion might contribute to the generation of mfl phenotype. However, snoH1 has little, if any, effect on mfl phenotypic rescue when over-expressed in mfl transgenic flies, either in the presence or in the absence of MFL overexpression. This is not surprising, given that each box H/ACA snoRNAs found in yeast, with the exception of snR30, is dispensable for viability. It will now be interesting to determine whether this type of gene organization is restricted to mfl or is shared by other members of this conserved gene family (Giordano, 1999).

DEVELOPMENTAL BIOLOGY

Embryonic

Nop60B RNA is found at high levels in nurse cells and in the oocyte, and is present throughout development. Nop60B protein is localized primarily to the nucleolus of interphase cells, and is absent from the chromosomes during mitosis (Phillips, 1998).

When the 2.0-kb mRNA was used on Northern blots, a probe derived from these two exons detects exclusively the 2.0-kb subform, specifically present in embryos and adult female RNAs. Hybridization of this probe to whole mount preparations of wild-type ovaries reveals that the female transcript accumulates in germ line cells from the early germarial until last oogenesis stages. The accumulation profile of both mfl mRNAs were followed during embryogenesis by developmental Northern blot analysis of carefully synchronized embryos. mRNAs are detected in very early, 0-2 h embryos. However, while the zygotic 1.8-kb mRNA persists at later stages, the level of female transcript drops subsequently, and becomes very low in 4-6 h embryos. This developmental pattern is very similar to that of other stable maternally supplied RNAs, which persist from early stages up to gastrulation (Giordano, 1999).

EFFECTS OF MUTATION

Nop60B mutants were generated and shown to be homozygous lethal. The Drosophila gene can rescue the lethal phenotype of yeast chf5 mutations, showing that the function of this protein has been conserved from yeast to Drosophila (Phillips, 1998).

The first minifly allele, mfl¹, was isolated in the course of a PZ-element mutagenesis screen on the second chromosome as a viable, recessive mutation causing a variety of phenotypic abnormalities. The mfl¹ pleiotropic phenotype included an extreme reduction of body size, developmental delay, essentially due to a 4-5-d prolongation of the larval life, defects in the abdominal cuticle, strong reduction in the length and thickness of abdominal bristles, and reduced female fertility. Most traits of the mfl¹ phenotype largely overlapped those caused by the Drosophila Minute or bobbed mutations that affect, respectively, the synthesis of ribosomal proteins, 5S, or 18S and 28S rRNAs. This similarity suggested for mfl a possible role in ribosome biogenesis, encouraging an attempt at the the molecular cloning of the gene (Giordano, 1999).

The mfl¹ mutation is caused by a single P-element insertion, which, by in situ hybridization of a P-specific probe to salivary gland polytene chromosomes of mfl¹ heterozygous larvae, was mapped on the chromosome arm 2R, at the 60B-60C polytene subdivisions boundary. Given that wild-type revertants could be recovered from dysgenic crosses after precise excision of the element, mfl¹ mutation appears to be directly caused by this single PZ insertion. Complementation analysis assigned the gene to the region covered by the Df(2R)Px4 deficiency. Among a number of P-induced lethal mutations deposited as part of the Berkeley Drosophila Genome Project, five mapped at the 60B-60C polytene subdivisions boundary. These mutations were all tested in a complementation analysis, by crossing each of them to mfl¹ heterozygous flies. Two lines, l(2)k05318 and l(2)k06308, yielded transheterozygous flies with a strong mfl phenotype at the expected ratio, leading to the conclusion that they belong to the mfl complementation group and represent lethal mfl alleles. Accordingly, these lines were renamed, respectively, mfl⁰⁵ and mfl⁰⁶. Previous cytological mapping by the Berkeley Drosophila Genome Project assigned these two mfl alleles to the polytene interval 60B11-C2. By lethal phase analysis it was observed that mfl⁰⁵ homozygotes die mainly as first instar larvae, while most of the mfl⁰⁶ animals die later, either as second or mainly as young third-instar larvae. Both mfl⁰⁵ and mfl⁰⁶ animals fail to increase their size as compared with their wild-type heterozygous siblings and survive for an additional 4-5 d as first or third instar larvae, respectively (Giordano, 1999).

Since a feature of the mfl¹ pleiotropic phenotype was represented by reduced female fertility, the structure of mutant ovaries was examined. Morphological abnormalities were often observed, with some of the egg chambers beginning to degenerate beyond approximately stage 7 of oogenesis. In the degenerating egg chambers, fragmented or condensed nurse cell nuclei with irregular shape are frequently found. These observations raise the possibility that apoptotic cell death may occur in mfl¹ abnormal ovaries. This possibility was investigated by staining egg chambers with acridine orange (AO). AO is a vital dye that is known to selectively stain apoptotic cells in insects and has successfully been used to study the distribution of apoptosis in Drosophila ovaries. Wild-type ovaries exhibit a diffuse green fluorescence, whereas highly fluorescent yellow spots are detected in mfl¹ degenerating egg chambers. These yellow spots are known to correspond to apoptotic, AO highly positive nuclei, thus confirming the occurrence of apoptosis in mfl¹ ovaries (Giordano, 1999).

As a consequence of the gonadic abnormalities observed, mfl¹ homozygous females lay a reduced number of mature eggs, and ~15% of the embryos produced fail to hatch. Such degenerating embryos show asynchronous and atypical development, invariantly accompanied by diffuse apoptotic cell death. Many mutations causing partial loss-of-function of vital genes interfere with the proper development of the egg, causing female sterility. Inadequate rate of protein synthesis is also known to affect Drosophila oogenesis, by slowing the level of yolk production and retarding egg chamber progression into vitellogenesis, beginning at stage 8. This effect is common to mutants unable to produce large amount of proteins, having reduced levels of either ribosomal proteins, 18S, 28S, or 5S rRNAs (Giordano, 1999).

When mfl mutants were checked for gene expression, they were found to have reduced levels of mfl mRNAs. While the viable, hypomorphic mfl¹ allele shows only a modest reduction, mfl expression is strongly disrupted in mfl⁰⁵ and mfl⁰⁶, the two alleles causing larval lethality. MFL protein accumulation strictly parallels the level of mfl mRNAs, so that it is strongly reduced in mfl⁰⁶ and nearly null in mfl⁰⁵. Remarkably, the developmental time at which lethality is achieved in these two mutants correlates well with MFL level since mfl⁰⁵ homozygotes die mainly as first instar larvae, while mfl⁰⁶ animals die as second or early third-instar larvae. Note that, considering the timing of persistence of maternal rRNA, mortality at the first instar is that which may be expected for mutations causing severe loss of function of a gene essential for rRNA processing. Taken together, all these data indicated that MFL level appear to be critical for Drosophila viability (Giordano, 1999).

Attempts were made to rescue mfl lethal phenotype by ectopically expressing MFL from the heat-inducible hsp70 promoter. mfl⁰⁵ and mfl⁰⁶ transgenic animals were then obtained and daily treated at 37°C for 30 min. These heat-shock conditions usually produce amounts of the ectopically expressed protein that largely exceed the wild-type level. However, in these experiments they produce a MFL level just comparable to that present in wild-type flies, even though the induced protein remains quite stable from 6 h to as long as 24 h from the heat-shock pulse. Nevertheless, the level of induced MFL is sufficient to allow mfl⁰⁵ and mfl⁰⁶ transformed animals to developed synchronously with their wild-type siblings and to show a normal increase in their size. Moreover, 30% of the mfl⁰⁵ and 80% of the mfl⁰⁶ transgenic animals develop up to the pupal stage, although these transgenic pupae all failed to eclose adult flies. A possible explanation for this partial rescue of the mfl mortality is that the level of ectopically expressed MFL may be inadequate with respect to the rate of protein synthesis required in specific cell types during metamorphosis. An alternative possibility is suggested by the observation that, in yeast, Cbf5p is required for the stability of other components of the H/ACA class of RNPs, such as Gar1p and box H/ACA snoRNAs (Lafontaine, 1998). It is thus possible that the MFL level reached under heat-shock conditions may not be constant and this affects the stability of other essential RNP components. However, since no member of the H/ACA class of RNPs has yet been described in Drosophila, this hypothesis cannot be tested at present (Giordano, 1999).

Nop60B is required in the maintenance of the germ line during spermatogenesis

The Nop60B gene is required in the maintenance of the germ line during spermatogenesis. Mutations impeding spermatogenesis often lead to reduced testis size in addition to male sterility. In general, defects that occur earlier in spermatogenesis cause a more severe size reduction. This appears to be because continued maintenance and division of germline stem cells is necessary to fill the testis with successive families of developing germ cells. Fly lines containing single P-element inserts were screened for male sterility and reduced testis size. This study focuses on sterile males with the profound testicular atrophy consistent with a defect in the maintenance of the stem cells. Two P{LacW} insertions, DL2011 and mM176, yielded such a phenotype and failed to complement each other. Precise excision of the P-element restored a wild type phenotype, demonstrating that the P insertion was responsible for the sterile phenotype. Flanking DNA was cloned and the insertion site was found to be in the 5' UTR of the Nop60B gene. Nop60B has been shown by sequence similarity to be a member of the TruB family of proteins, all of which show homology to Escherichia coli TruB, a tRNA pseudouridine synthase (Phillips, 1998). Several 'imprecise' excisions were analyzed; this analysis yielded lethal and semiviable alleles, as well as several viable but sterile alleles, such as nop60^BDL4, which showed atrophic testes similar to DL2011 (Kauffman, 2003).

Testes from nop60^BDL4 mutants are severely reduced in size compared with a wild-type testis, suggesting a significant reduction in the number of germline cells. This was verified by immunostaining with mAB-1B1, which marks the fusome, a germ-line-specific cytoskeletal organelle. In a wild type testis, germ-line stem cells adjacent to the hub exhibit dot fusomes, while later stage germ cells at a distance from the hub show progressively more branched fusomes, reflecting mitotic amplification of the gonial cells. Testes from nop60^BDL4 mutants exhibited a near absence of fusome signal (Kauffman, 2003).

To verify that the testis phenotype was due to the insertion of the P element into the Nop60B gene, rescue experiments were performed using a HS-Nop60B transgenic line (Giordano, 1999). Daily heat shocks began 0-1 day after egg laying (AEL) and continued until adult flies eclosed. Testes were then dissected, fixed, and immunostained with mAB-1B1. nop60^BDL4 homozygotes carrying the rescue construct showed dramatic heat shock-dependent rescue of testicular atrophy, as shown by the reappearance of fusomes. Ninety-one percent (91%) of testes from young males heat shocked until the day of dissection showed more than 10 branched fusomes per testis. Importantly, germ-line stem cells were also rescued, as judged by the presence of germ cells with dot fusomes adjacent to the hub. In contrast, only 17% of testes from age-matched nop60^BDL4 flies raised without heat shock showed any evidence of germ cells, while 75% showed none. The rescue confirms that the testicular atrophy phenotype is due to a defect in the Nop60B gene. Thus, Nop60B is required in some manner for the maintenance of the germ line during spermatogenesis (Kauffman, 2003).

A panel of molecular markers was used to address the nature of the nop60^BDL4 phenotype at the cellular level and ascertain the effect of this mutation on the various cell populations in the testis. Using such markers, it was found that nop60^BDL4 testes showed a dramatic reduction in the number of germ cells relative to wild type, without exhibiting a similar loss of somatic cells. The fusome stain first suggested a strong germ cell phenotype. This was verified by testing for expression of the germ-line-specific protein Vasa. No germ cells are apparent in the mutant testis, as judged by the lack of signal. The lack of germ cells included an apparent lack of germ-line stem cells. This was confirmed by taking advantage of lacZ enhancer traps that mark specific cell types and stages during spermatogenesis. Of particular use in this analysis was the recovery of Nop60B alleles, such as nop60^BDL4, which lost the ability to express lacZ, allowing analysis of the expression of lacZ markers that were introduced into nop60^BDL4 flies. In wild-type testes, enhancer trap line M5-4 marks the hub, germ-line stem cells, and their immediate daughters, the gonialblast cells. In mutant testes, hub cells were present, as judged by the presence of groups of M5-4-expressing cells, although it was noted that these cells had an elongate appearance, which is similar to the disorganization in hub cells observed in agametic testes. Another similarity between agametic and nop60^BDL4 mutant testes is that hub cells accumulate Fasciclin III protein to levels lower than those observed in wild type. Although hub cells were observed, usually no evidence for germ-line stem cells or their gonialblast progeny were found. Occasionally, a few lacZ-expressing germ cells were observed, but these were some distance from the hub. It was not clear whether these cells were wandering gonialblasts or germ-line stem cells. There are currently no markers with expression restricted solely to germ-line stem cells. It is concluded from these analyses that testes are atrophic due to severe loss of germ-line cells, including the germ-line stem cells (Kauffman, 2003).

Despite the loss of the germ cell population, the cyst cells, which derive from somatic stem cells, were observed in mutant testes, as judged by both an enhancer trap marker and the expression of Eyes Absent (Eya). In wild type, enhancer trap S2-11, which marks hub and early-stage cyst cells, shows an organized hub and an appropriately robust early-stage cyst cell population. In mutant testes, hub cells are apparent, and exhibit a similar loss of organization as that observed with enhancer trap M5-4 and in agametic testes. However, unlike the germ cell population, which is quite depleted, significant numbers of cyst cells are observed. That there is little effect on the cyst cell population was confirmed by examining the expression of Eya, a cyst cell-specific marker. Eya expression is first induced in cyst cells associated with midamplification stage gonia, approximately at the four- to eight-cell stage. Thus, Eya expression is not only specific to the cyst cell lineage, but its expression can be taken as a measure that the cyst cells have progressed to a certain point in their developmental program. Indeed, in mutant testes, large numbers of Eya-positive cyst cells accumulate. This confirms that there is not as large an effect on the somatic cells, as compared with the germ line. It was not possible to assay directly for cyst progenitor cells (the somatic stem cells), since the only marker for these cells is a P enhancer trap that resides on the CyO balancer chromosome, which cannot be used in nop60^BDL4 homozygotes. Nevertheless, it is clear from these data that, in nop60^BDL4 mutant testes, the behavior of the somatic stem cell lineage is not affected to the same degree as is the germ-line stem cell population and its descendants. Specific deletion of germ cells, achieved in agametic testes, leads to a loss of hub organization, expansion in the number of hub cells, and continued cyst cell proliferation. Thus, it appears that, in nop60^BDL4 testes, the primary defect may be loss of the germ-line cells, leading to associated changes in somatic cell behavior (Kauffman, 2003).

Next addressed was the question of whether the Nop60B gene product is required for the maintenance of the germ-line stem cell population. Three different approaches to this question indicate that continued Nop60B gene function is necessary for the maintenance of the stem cell lineage (Kauffman, 2003).

It is concluded that the testicular atrophy observed in Nop60B gene mutation is due to an almost complete lack of germ cells in adult flies. The somatic cyst cell population, however, is not significantly reduced. By several criteria, the germ-line stem cells, at least some of which are initially specified, are prematurely lost, accounting for the virtual loss of the germ-line lineage in adult testes. In addition, the requirement for Nop60B is intrinsic to the germ-line; removing Nop60B activity leads to loss of germline stem cells and death of gonial cells. This implicates the Nop60B gene in the maintenance of the germ line during spermatogenesis (Kauffman, 2003).

In principle, it is possible that Nop60B plays a special role in germ cells, or in germ-line stem cells, which would explain the loss of this lineage in the hypomorphic mutants investigated here. An explanation such as this would justify screening for germ-line loss mutants on a large scale in order to identify genes governing the behavior of stem cell lineages. Alternatively, and more likely in this particular case, the hypomorphic nature of the Nop60B alleles may account for this apparent requirement solely in a renewing lineage. For instance, there may be enough gene product both from maternal stores and residual zygotic expression to support larval life and metamorphosis, but not enough to maintain the germ-line lineage in spermatogenesis. An explanation such as this makes one hesitate in trying to identify interesting genes governing the behavior of stem cell lineages from among mutants showing germ cell loss phenotypes (Kauffman, 2003).

Indeed, Nop60B encodes a protein likely involved centrally in cellular metabolism. It contains homology to a class of pseudouridine synthases, and, although this activity has yet to be directly demonstrated, the Nop60B protein is localized to the nucleolus, the site of rRNA processing, in all cell populations (Phillips, 1998), and a previous analysis of null mutant larvae strongly suggests defects in rRNA processing and pseudouridinylation (Giordano, 1999). It is not surprising then that strong mutations in this gene are not stem cell- or germ-line-specific, but are rather lethal to the organism at a very early stage. That hypomorphic alleles have a dramatic effect on the germline is likely due to the need to sustain proliferation in this adult tissue, or more particularly, sustain high rates of protein synthesis. This is a similar conclusion to that arrived at previously in order to explain the small body size phenotype of the 'mini-fly' class of Nop60B alleles (Giordano, 1999). The hypomorphic alleles used on this study did not exhibit a mini-fly phenotype, and, thus, do not appear to be generally compromising the fly. This has afforded the ability to investigate the requirement for Nop60B in tissue homeostasis and stem cell function, a role which might relate to that affected in a human disease (Kauffman, 2003).

Within the testis, the data suggest that the requirement for Nop60B gene product may be highest in the germ-line stem cells. The testes from most rescued hypomorphic Nop60B mutant flies initially have a functioning germline, and the germ cells are lost if the flies are aged without continued production of rescuing Nop60B activity. The fact that a few germ-line stem cells are apparent in the third larval instar gonad, but not in adults, also supports the proposition that stem cells are initially specified but cannot be maintained. Interestingly, it was also found that the somatic stem cells and their daughter cyst cells appear to be less affected than the germ line under these hypomorphic conditions. It is possible that the P insertion affects the expression of Nop60B in the germ line more than in the somatic lineage. However, the current understanding of the gene structure of Nop60B provides no easy explanation for this, such as a testis-specific promoter (Giordano, 1999; Phillips, 1998). Alternatively, the data suggest that there is a higher requirement for Nop60B gene product for the steady-state operation of the germ-line stem cells. Although it has been shown that removing Nop60B function completely from the germ-line stem cells leads to their loss, similar mosaic analysis could not be performd on the somatic stem cell population because the somatic markers are not as robust (Kauffman, 2003).

REFERENCES

Search PubMed for articles about Drosophila Nucleolar protein at 60B

Cadwell, C., et al. (1997). The yeast nucleolar protein Cbf5p is involved in rRNA biosynthesis and interacts genetically with the RNA polymerase I transcription factor RRN3. Mol. Cell Biol. 17: 6175-6183. 9315678

Dez, C., Henras, A., Faucon, B., Lafontaine, D., Caizergues-Ferrer, M. and Henry, Y. (2001). Stable expression in yeast of the mature form of human telomerase RNA depends on its association with the box H/ACA small nucleolar RNP proteins Cbf5p, Nhp2p and Nop10p. Nucleic Acids Res. 29(3): 598-603. 11160879

Giordano, E., Peluso, I., Senger, S. and Furia, M. (1999). minifly, a Drosophila gene required for ribosome biogenesis. J. Cell Biol. 144: 1123-1133. 10087258

Heiss, N. S., et al. (1998). X-linked dyskeratosis congenita is caused by mutations in a highly conserved gene with putative nucleolar functions. Nat. Genet. 19: 32-38. 9590285

Jiang, W., et al. (1993). An essential yeast protein, CBF5p, binds in vitro to centromeres and microtubules. Mol. Cell Biol. 13: 4884-4893. 8336724

Kauffman, T., Tran, J. and DiNardo, S. (2003). Mutations in Nop60B, the Drosophila homolog of human Dyskeratosis congenita 1, affect the maintenance of the germ-line stem cell lineage during spermatogenesis. Dev. Biol. 253: 189-199. 12645924

Kavli, B., et al. (1996). Excision of cytosine and thymine from DNA by mutants of human uracil-DNA glycosylase. EMBO J. 15: 3442-3447. 8670846

Kendall. A., Hull, M. W., Bertrand, E., Good, P. D., Singer, R. H. and Engelke, D. R. (2000). A CBF5 mutation that disrupts nucleolar localization of early tRNA biosynthesis in yeast also suppresses tRNA gene-mediated transcriptional silencing. Proc. Natl. Acad. Sci. 97(24): 13108-13. 11069303

Lafontaine, D. L. J., et al. (1998). The box H + ACA snoRNAs carry Cbf5p, the putative rRNA pseudouridine synthase. Genes Dev. 12: 527-537. 9472021

Luzzatto, L., and Karadimitris, A. (1998). Dyskeratosis and ribosomal rebellion. Nat. Genet. 19: 6-7. 9590276

Meier, U.T., and Blobel, G. (1994). NAP57, a mammalian nucleolar protein with a putative homolog in yeast and bacteria. J. Cell Biol. 127: 1505-1514. 7798307

Mitchell, J. R., Wood, E. and Collins, K. (1999). A telomerase component is defective in the human disease dyskeratosis congenita Nature 402: 551-555. 10591218

Phillips, B., Billin, A. N., Cadwell, C., Buchholz, R., Erickson, C., Merriam, J. R., Carbon, J. and Poole, S. J. (1998). The Nop60B gene of Drosophila encodes an essential nucleolar protein that functions in yeast. Molec. Gen. Genet. 260(1): 20-29. 9829824

Slupphaug, G., et al. (1996). A nucleotide-flipping mechanism from the structure of human uracil-DNA glycosylase bound to DNA. Nature. 384: 87-92. 8900285

Smith, C.M., and J.A. Steitz. 1997. Sno storm in the nucleolus: new roles for myriad small RNPs. Cell 89: 669-672. 9182752

Watkins, N. J., et al. (1998). Cbf5p, a potential pseudouridine synthase, and Nhp2p, a putative RNA-binding protein, are present together with Gar1p in all H BOX/ACA-motif snoRNPs and constitute a common bipartite structure. RNA. 4(12): 1549-68. 9848653

Zebarjadian, Y., King, T., Fournier, M. J., Clarke, L. and Carbon, J. (1999). Point mutations in yeast CBF5 can abolish in vivo pseudouridylation of rRNA. Mol Cell Biol. 19(11): 7461-72. 10523634

date revised: 30 March 2003

Home page: The Interactive Fly © 2017 Thomas Brody, Ph.D.