Contrasting patterns of evolutionary constraint and novelty revealed by comparative sperm proteomic analysis in Lepidoptera

Background: Rapid evolution is a hallmark of reproductive genetic systems and arises through the combined processes of sequence divergence, gene gain and loss, and changes in gene and protein expression. While studies aiming to disentangle the molecular ramifications of these processes are progressing, we still know little about the genetic basis of evolutionary transitions in reproductive systems. Here we conduct the first comparative analysis of sperm proteomes in Lepidoptera, a group that exhibits dichotomous spermatogenesis, in which males produce a functional fertilization-competent sperm (eupyrene) and an incompetent sperm morph lacking nuclear DNA (apyrene). Through the integrated application of evolutionary proteomics and genomics, we characterize the genomic patterns potentially associated with the origination and evolution of this unique spermatogenic process and assess the importance of genetic novelty in Lepidopteran sperm biology. Results: Comparison of the newly characterized Monarch butterfly (Danaus plexippus) sperm proteome to those of the Carolina sphinx moth (Manduca sexta) and the fruit fly (Drosophila melanogaster) demonstrated conservation at the level of protein abundance and post-translational modification within Lepidoptera. In contrast, comparative genomic analyses across insects reveals significant divergence at two levels that differentiate the genetic architecture of sperm in Lepidoptera from other insects. First, a significant reduction in orthology among Monarch sperm genes relative to the remainder of the genome in non-Lepidopteran insect species was observed. Second, a substantial number of sperm proteins were found to be specific to Lepidoptera, in that they lack detectable homology to the genomes of more distantly related insects. Lastly, the functional importance of Lepidoptera specific sperm proteins is broadly supported by their increased abundance relative to proteins conserved across insects. Conclusions: Our results identify a burst of genetic novelty amongst sperm proteins that may be associated with the origin of heteromorphic spermatogenesis in ancestral Lepidoptera and/or the subsequent evolution of this system. This pattern of genomic diversification is distinct from the remainder of the genome and thus suggests that this transition has had a marked impact on lepidopteran genome evolution. The identification of abundant sperm proteins unique to Lepidoptera, including proteins distinct between specific lineages, will accelerate future functional studies aiming to understand the developmental origin of dichotomous spermatogenesis and the functional diversification of the fertilization incompetent apyrene sperm morph

Gene processing control loops suggested by sequencing, splicing, and RNA folding

Abstract Background Small RNAs are known to regulate diverse gene expression processes including translation, transcription, and splicing. Among small RNAs, the microRNAs (miRNAs) of 17 to 27 nucleotides (nts) undergo biogeneses including primary transcription, RNA excision and folding, nuclear export, cytoplasmic processing, and then bioactivity as regulatory agents. We propose that analogous hairpins from RNA molecules that function as part of the spliceosome might also be the source of small, regulatory RNAs (somewhat smaller than miRNAs). Results Deep sequencing technology has enabled discovery of a novel 16-nt RNA sequence in total RNA from human brain that we propose is derived from RNU1, an RNA component of spliceosome assembly. Bioinformatic alignments compel inquiring whether the novel 16-nt sequence or its precursor have a regulatory function as well as determining aspects of how processing intersects with the miRNA biogenesis pathway. Specifically, our preliminary in silico investigations reveal the sequence could regulate splicing factor Arg/Ser rich 1 (SFRS1), a gene coding an essential protein component of the spliceosome. All 16-base source sequences in the UCSC Human Genome Browser are within the 14 instances of RNU1 genes listed in wgEncodeGencodeAutoV3. Furthermore, 10 of the 14 instances of the sequence are also within a common 28-nt hairpin-forming subsequence of RNU1. Conclusions An abundant 16-nt RNA sequence is sourced from a spliceosomal RNA, lies in a stem of a predicted RNA hairpin, and includes reverse complements of subsequences of the 3'UTR of a gene coding for a spliceosome protein. Thus RNU1 could function both as a component of spliceosome assembly and as inhibitor of production of the essential, spliceosome protein coded by SFRS1. Beyond this example, a general procedure is needed for systematic discovery of multiple alignments of sequencing, splicing, and RNA folding data

High-Throughput Functional MicroRNAs Profiling by Recombinant AAV-Based MicroRNA Sensor Arrays

BACKGROUND: microRNAs (miRNAs) are small and non-coding RNAs which play critical roles in physiological and pathological processes. A number of methods have been established to detect and quantify miRNA expression. However, method for high-throughput miRNA function detection is still lacking. PRINCIPAL FINDINGS: We describe an adeno-associated virus (AAV) vector-based microRNA (miRNA) sensor (Asensor) array for high-throughput functional miRNA profiling. Each Asensor contains a Gaussia luciferase (Gluc) and a firefly luciferase (Fluc) expression cassette to sense functional miRNA and to serve as an internal control respectively. Using this array, we acquired functional profiles of 115 miRNAs for 12 cell lines and found "functional miRNA signatures" for several specific cell lines. The activities of specific miRNAs including the let-7 family, miR-17-92 cluster, miR-221, and miR-222 in HEK 293 cells were compared with their expression levels determined by quantitative reverse transcriptase polymerase chain reaction (QRT-PCR). We also demonstrate two other practical applications of the array, including a comparison of the miRNA activity between HEK293 and HEK293T cells and the ability to monitor miRNA activity changes in K562 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA). CONCLUSIONS/SIGNIFICANCE: Our approach has potential applications in the identification of cell types, the characterization of biological and pathological processes, and the evaluation of responses to interventions

The patriotism of gentlemen with red hair: European Jews and the liberal state, 1789–1939

European Jewish history from 1789–1939 supports the view that construction of national identities even in secular liberal states was determined not only by modern considerations alone but also by ancient patterns of thought, behaviour and prejudice. Emancipation stimulated unprecedented patriotism, especially in wartime, as Jews strove to prove loyalty to their countries of citizenship. During World War I, even Zionists split along national lines, as did families and friends. Jewish patriotism was interchangeable with nationalism inasmuch as Jews identified themselves with national cultures. Although emancipation implied acceptance and an end to anti-Jewish prejudice in the modern liberal state, the kaleidoscopic variety of Jewish patriotism throughout Europe inadvertently undermined the idea of national identity and often provoked anti-Semitism. Even as loyal citizens of separate states, the Jews, however scattered, disunited and diverse, were made to feel, often unwillingly, that they were one people in exile