Evidence for post-transcriptional regulation of clustered microRNAs in Drosophila

BACKGROUND: MicroRNAs (miRNA) are short 21-23nt RNAs capable of inhibiting translation of complementary target messenger RNAs. Almost half of D. melanogaster miRNA genes are grouped in genomic clusters. RESULTS: The peculiarities of the expression of clustered miRNAs were studied using publicly available libraries of sequenced small RNAs from different Drosophila tissues. We have shown that although miRNAs from almost all clusters have similar tissue expression profiles (coordinated clusters), some clusters contain miRNAs with uncoordinated expression profiles. The predicted transcription start sites (TSSs) of such clusters are located upstream of the first miRNA, but no TSSs are found within the clusters. The expression profiles of miR and miR* sequences in uncoordinated clustered miRNAs do not correlate while their profiles from the coordinated clustered miRNAs are similar. CONCLUSIONS: The presence of exclusively upstream promoters in miRNA clusters containing uncoordinated miRNAs means that the clusters are transcribed as single transcription units. The difference of tissue expression profiles of uncoordinated miRNAs and the corresponding miRs* suggests a post-transcriptional regulation of their processing or stability. [KEYWORDS: Animals, Drosophila/ genetics, Gene Expression Profiling, MicroRNAs/ genetics/ metabolism, Multigene Family, Promoter Regions, Genetic, RNA Processing, Post-Transcriptional, Transcription Initiation Site]

The differences between Cis-and Trans-Gene inactivation caused by heterochromatin in Drosophila

Position-effect variegation (PEV) is the epigenetic disruption of gene expression near the de novo-formed euchromatin-heterochromatin border. Heterochromatic cis-inactivation may be accompanied by the trans-inactivation of genes on a normal homologous chromosome in trans-heterozygous combination with a PEV-inducing rearrangement. We characterize a new genetic system, inversion In(2)A4, demonstrating cis-acting PEV as well as trans-inactivation of the reporter transgenes on the homologous nonrearranged chromosome. The cis-effect of heterochromatin in the inversion results not only in repression but also in activation of genes, and it varies at different developmental stages. While cis-actions affect only a few juxtaposed genes, trans-inactivation is observed in a 500-kb region and demonstrates а nonuniform pattern of repression with intermingled regions where no transgene repression occurs. There is no repression around the histone gene cluster and in some other euchromatic sites. trans-Inactivation is accompanied by dragging of euchromatic regions into the heterochromatic compartment, but the histone gene cluster, located in the middle of the trans-inactivated region, was shown to be evicted from the heterochromatin. We demonstrate that trans-inactivation is followed by de novo HP1a accumulation in the affected transgene; trans-inactivation is specifically favored by the chromatin remodeler SAYP and prevented by Argonaute AGO2

Repeat-associated siRNAs cause chromatin silencing of retrotransposons in the Drosophila melanogaster germline

Silencing of genomic repeats, including transposable elements, in Drosophila melanogaster is mediated by repeat-associated short interfering RNAs (rasiRNAs) interacting with proteins of the Piwi subfamily. rasiRNA-based silencing is thought to be mechanistically distinct from both the RNA interference and microRNA pathways. We show that the amount of rasiRNAs of a wide range of retroelements is drastically reduced in ovaries and testes of flies carrying a mutation in the spn-E gene. To address the mechanism of rasiRNA-dependent silencing of retrotransposons, we monitored their chromatin state in ovaries and somatic tissues. This revealed that the spn-E mutation causes chromatin opening of retroelements in ovaries, resulting in an increase in histone H3 K4 dimethylation and a decrease in histone H3 K9 di/trimethylation. The strongest chromatin changes have been detected for telomeric HeT-A elements that correlates with the most dramatic increase of their transcript level, compared to other mobile elements. The spn-E mutation also causes depletion of HP1 content in the chromatin of transposable elements, especially along HeT-A arrays. We also show that mutations in the genes controlling the rasiRNA pathway cause no derepression of the same retrotransposons in somatic tissues. Our results provide evidence that germinal Piwi-associated short RNAs induce chromatin modifications of their targets

Peculiarities of piRNA-mediated post-transcriptional silencing of Stellate repeats in testes of Drosophila melanogaster

Silencing of Stellate genes in Drosophila melanogaster testes is caused by antisense piRNAs produced as a result of transcription of homologous Suppressor of Stellate (Su(Ste)) repeats. Mechanism of piRNA-dependent Stellate repression remains poorly understood. Here, we show that deletion of Su(Ste) suppressors causes accumulation of spliced, but not nonspliced Stellate transcripts both in the nucleus and cytoplasm, revealing post-transcriptional degradation of Stellate RNA as the predominant mechanism of silencing. We found a significant amount of Su(Ste) piRNAs and piRNA-interacting protein Aubergine (Aub) in the nuclear fraction. Immunostaining of isolated nuclei revealed co-localization of a portion of cellular Aub with the nuclear lamina. We suggest that the piRNA–Aub complex is potentially able to perform Stellate silencing in the cell nucleus. Also, we revealed that the level of the Stellate protein in Su(Ste)-deficient testes is increased much more dramatically than the Stellate mRNA level. Similarly, Su(Ste) repeats deletion exerts an insignificant effect on mRNA abundance of the Ste-lacZ reporter, but causes a drastic increase of β-gal activity. In cell culture, exogenous Su(Ste) dsRNA dramatically decreases β-gal activity of hsp70-Ste-lacZ construct, but not its mRNA level. We suggest that piRNAs, similarly to siRNAs, degrade only unmasked transcripts, which are accessible for translation

Expansion and Evolution of the X-Linked Testis Specific Multigene Families in the melanogaster Species Subgroup

The testis specific X-linked genes whose evolution is traced here in the melanogaster species subgroup are thought to undergo fast rate of diversification. The CK2ßtes and NACβtes gene families encode the diverged regulatory β-subunits of protein kinase CK2 and the homologs of β-subunit of nascent peptide associated complex, respectively. We annotated the CK2βtes-like genes related to CK2ßtes family in the D. simulans and D. sechellia genomes. The ancestor CK2βtes-like genes preserved in D. simulans and D. sechellia are considered to be intermediates in the emergence of the D. melanogaster specific Stellate genes related to the CK2ßtes family. The CK2ßtes-like genes are more similar to the unique autosomal CK2ßtes gene than to Stellates, taking into account their peculiarities of polymorphism. The formation of a variant the CK2ßtes gene Stellate in D. melanogaster as a result of illegitimate recombination between a NACßtes promoter and a distinct polymorphic variant of CK2ßtes-like ancestor copy was traced. We found a close nonrandom proximity between the dispersed defective copies of DINE-1 transposons, the members of Helitron family, and the CK2βtes and NACβtes genes, suggesting an involvement of DINE-1 elements in duplication and amplification of these genes

Molecular Evolution of Two Paralogous Tandemly Repeated Heterochromatic Gene Clusters Linked to the X and Y Chromosomes of Drosophila melanogaster

Here we report the peculiarities of molecular evolution and divergence of paralogous heterochromatic clusters of the testis- expressed X-linked Stellate and Y-linked Su(Ste) tandem repeats. It was suggested that Stellate and Su(Ste) clusters affecting male fertility are the amplified derivatives of the unique euchromatic gene βCK2tes encoding the putative testis-specific β-subunit of protein kinase CK2. The putative Su(Ste)-like evolutionary intermediate was detected on the Y chromosome as an orphon outside of the Su(Ste) cluster. The orphon shows extensive homology to the Su(Ste) repeat, but contains several Stellate-like diagnostic nucleotide substitutions, as well as a 10-bp insertion and a 3′ splice site of the first intron typical of the Stellate unit. The orphon looks like a pseudogene carrying a drastically damaged Su(Ste) open reading frame (ORF). The putative Su(Ste) ORF, as compared with the Stellate one, carries numerous synonymous substitutions leading to the major codon preference. We conclude that Su(Ste) ORFs evolved on the Y chromosome under the pressure of translational selection. Direct sequencing shows that the efficiency of concerted evolution between adjacent repeats is 5–10 times as high in the Stellate heterochromatic cluster on the X chromosome as that in the Y-linked Su(Ste) cluster, judging by the frequencies of nucleotide substitutions and single-nucleotide deletions

Molecular Evolution of Two Paralogous Tandemly Repeated Heterochromatic Gene Clusters Linked to the X and Y Chromosomes of Drosophila melanogaster

Here we report the peculiarities of molecular evolution and divergence of paralogous heterochromatic clusters of the testis- expressed X-linked Stellate and Y-linked Su(Ste) tandem repeats. It was suggested that Stellate and Su(Ste) clusters affecting male fertility are the amplified derivatives of the unique euchromatic gene βCK2tes encoding the putative testis-specific β-subunit of protein kinase CK2. The putative Su(Ste)-like evolutionary intermediate was detected on the Y chromosome as an orphon outside of the Su(Ste) cluster. The orphon shows extensive homology to the Su(Ste) repeat, but contains several Stellate-like diagnostic nucleotide substitutions, as well as a 10-bp insertion and a 3′ splice site of the first intron typical of the Stellate unit. The orphon looks like a pseudogene carrying a drastically damaged Su(Ste) open reading frame (ORF). The putative Su(Ste) ORF, as compared with the Stellate one, carries numerous synonymous substitutions leading to the major codon preference. We conclude that Su(Ste) ORFs evolved on the Y chromosome under the pressure of translational selection. Direct sequencing shows that the efficiency of concerted evolution between adjacent repeats is 5–10 times as high in the Stellate heterochromatic cluster on the X chromosome as that in the Y-linked Su(Ste) cluster, judging by the frequencies of nucleotide substitutions and single-nucleotide deletions