The functional significance of ribosome heterogeneity in development and differentiation is relatively unexplored. We present the first in vivo evidence of ribosome heterogeneity playing a role in ...specific mRNA translation in a multicellular eukaryote. Eukaryotic-specific ribosomal protein paralogues eRpL22 and eRpL22-like are essential in development and required for sperm maturation and fertility in
. eRpL22 and eRpL22-like roles in spermatogenesis are not completely interchangeable. Flies depleted of eRpL22 and rescued by eRpL22-like overexpression have reduced fertility, confirming that eRpL22-like cannot substitute fully for eRpL22 function, and that paralogues have functionally distinct roles, not yet defined. We investigated the hypothesis that specific RNAs differentially associate with eRpL22 or eRpL22-like ribosomes, thereby establishing distinct ribosomal roles. RNA-seq identified 12,051 transcripts (mRNAs/noncoding RNAs) with 50% being enriched on specific polysome types. Analysis of ∼10% of the most abundant mRNAs suggests ribosome specialization for translating groups of mRNAs expressed at specific stages of spermatogenesis. Further, we show enrichment of "model" eRpL22-like polysome-associated testis mRNAs can occur outside the germline within S2 cells transfected with eRpL22-like, indicating that germline-specific factors are not required for selective translation. This study reveals specialized roles in translation for eRpL22 and eRpL22-like ribosomes in germline differentiation.
Abstract
Comparative analyses of mycobacteriophage genomes reveals extensive genetic diversity in genome organization and gene content, contributing to widespread mosaicism. We previously reported ...that the prophage of mycobacteriophage Butters (cluster N) provides defense against infection by Island3 (subcluster I1). To explore the anti-Island3 defense mechanism, we attempted to isolate Island3 defense escape mutants on a Butters lysogen, but only uncovered phages with recombinant genomes comprised of regions of Butters and Island3 arranged from left arm to right arm as Butters-Island3-Butters (BIBs). Recombination occurs within two distinct homologous regions that encompass lysin A, lysin B, and holin genes in one segment, and RecE and RecT genes in the other. Structural genes of mosaic BIB genomes are contributed by Butters while the immunity cassette is derived from Island3. Consequently, BIBs are morphologically identical to Butters (as shown by transmission electron microscopy) but are homoimmune with Island3. Recombinant phages overcome antiphage defense and silencing of the lytic cycle. We leverage this observation to propose a stratagem to generate novel phages for potential therapeutic use.
Graphical Abstract
Graphical Abstract
In silico comparative analysis of mycobacteriophage genomes reveals extensive genetic diversity and widespread genome mosaicism. Mohammed et al., describe a novel mosaic recombinant phage named BIB (Butters-Island3-Butters) produced in situ by homologous recombination between the Mycobacteriophage Butters (cluster N) prophage within a lysogen and the invading Island3 (subcluster I1) genome. Mycobacteriophage Butters prophage-encoded factors block lytic infection of heterotypic Mycobacteriophage Island3 but the recombinant BIB genome contains genetic modules that allow escape from prophage-mediated defense and repressor-mediated lytic repression.
Background
Differences in core or tissue‐specific ribosomal protein (Rp) composition within ribosomes contribute to ribosome heterogeneity and functional variability. Yet, the degree to which ...ribosome heterogeneity modulates development is unknown. The Drosophila melanogaster eRpL22 family contains structurally diverse paralogues, eRpL22 and eRpL22‐like. Unlike ubiquitously expressed eRpL22, eRpL22‐like expression is tissue‐specific, notably within the male germline and the eye. We investigated expression within the developing eye to uncover tissue/cell types where specific paralogue roles might be defined.
Results
Immunohistochemistry analysis confirms ubiquitous eRpL22 expression throughout eye development. In larvae, eRpL22‐like is ubiquitously expressed, but highly enriched in the peripodial epithelium (PE). In early pupae, eRpL22‐like is broadly distributed in multiple cell types, but later, is primarily enriched in interommatidial hair cells (IoHC). Adult patterns include the ring of accessory cells around ommatidia. Adult retinae IoHC patterning phenotypes (shown by scanning electron microscopy) may be linked to RNAi‐mediated eRpL22‐like depletion within larval PE. Immunoblots and polysome profile analyses show multiple variants of eRpL22‐like across development, with the variant at the expected molecular mass co‐sedimenting with active ribosomes.
Conclusion
Our data reveal differential patterns of eRpL22‐like expression relative to eRpL22 and suggest a specific role for eRpL22‐like in developmental patterning of the eye.
Key Findings
Ribosomal protein paralogues eRpL22 and eRpL22‐like are co‐expressed in the developing eye.
eRpL22‐like expression patterns change depending on developmental stage.
eRpL22‐like is enriched in the larval peripodial epithelium (PE), the pupal interommatidial hair cell (IoHC), and the ring of accessory cells in adult ommatidia.
IoHC orientation and structural phenotypes in adult retinae may be associated with RNAi‐mediated depletion of eRpL22‐like within the PE of third instar larvae, suggesting a role for eRpL22‐like in IoHC patterning.
Different molecular variants of eRpL22‐like are expressed at different development stages, but not all variants associate with ribosomal components.
Duplicated ribosomal protein (RP) genes in the Drosophila melanogaster eRpL22 family encode structurally-divergent and differentially-expressed rRNA-binding RPs. eRpL22 is expressed ubiquitously and ...eRpL22-like expression is tissue-restricted with highest levels in the adult male germline. We explored paralogue functional equivalence using the GAL4-UAS system for paralogue knockdown or overexpression and a conditional eRpL22-like knockout in a heat- shock flippase/FRT line. Ubiquitous eRpL22 knockdown with Actin-GAL4 resulted in embryonic lethality, confirming eRpL22 essentiality. eRpL22-like knockdown (60%) was insufficient to cause lethality; yet, conditional eRpL22-like knockout at one hour following egg deposition caused lethality within each developmental stage. Therefore, each paralogue is essential. Variation in timing of heat-shock-induced eRpL22-like knockout highlighted early embryogenesis as the critical period where eRpL22-like expression (not compensated for by eRpL22) is required for normal development of several organ systems, including testis development and subsequent sperm production. To determine if eRpL22-like can substitute for eRpL22, we used Actin-GAL4 for ubiquitous eRpL22 knockdown and eRpL22-like-FLAG (or FLAG-eRpL22: control) overexpression. Emergence of adults demonstrated that ubiquitous eRpL22-like-FLAG or FLAG-eRpL22 expression eliminates embryonic lethality resulting from eRpL22 depletion. Adults rescued by eRpL22-like-FLAG (but not by FLAG-eRpL22) overexpression had reduced fertility and longevity. We conclude that eRpL22 paralogue roles are not completely interchangeable and include functionally-diverse roles in development and spermatogenesis. Testis-specific paralogue knockdown revealed molecular phenotypes, including increases in eRpL22 protein and mRNA levels following eRpL22-like depletion, implicating a negative crosstalk mechanism regulating eRpL22 expression. Paralogue depletion unmasked mechanisms, yet to be defined that impact paralogue co-expression within germ cells.
Several ribosomal protein families contain paralogues whose roles may be equivalent or specialized to include extra-ribosomal functions. RpL22e family members rpL22 and rpL22-like are differentially ...expressed in Drosophila melanogaster: rpL22-like mRNA is gonad specific whereas rpL22 is expressed ubiquitously, suggesting distinctive paralogue functions. To determine if RpL22-like has a divergent role in gonads, rpL22-like expression was analysed by qRT-PCR and western blots, respectively, showing enrichment of rpL22-like mRNA and a 34 kDa (predicted) protein in testis, but not in ovary. Immunohistochemistry of the reproductive tract corroborated testis-specific expression. RpL22-like detection in 80S/polysome fractions from males establishes a role for this tissue-specific paralogue as a ribosomal component. Unpredictably, expression profiles revealed a low abundant, alternative mRNA variant (designated 'rpL22-like short') that would encode a novel protein lacking the C-terminal ribosomal protein signature but retaining part of the N-terminal domain. This variant results from splicing of a retained intron (defined by non-canonical splice sites) within rpL22-like mRNA. Polysome association and detection of a low abundant 13.5 kDa (predicted) protein in testis extracts suggests variant mRNA translation. Collectively, our data show that alternative splicing of rpL22-like generates structurally distinct protein products: ribosomal component RpL22-like and a novel protein with a role distinct from RpL22-like.
A diverse set of prophage-mediated mechanisms protecting bacterial hosts from infection has been recently uncovered within cluster N mycobacteriophages isolated on the host,
mc
155. In that context, ...we unveil a novel defense mechanism in cluster N prophage Butters. By using bioinformatics analyses, phage plating efficiency experiments, microscopy, and immunoprecipitation assays, we show that Butters genes located in the central region of the genome play a key role in the defense against heterotypic viral attack. Our study suggests that a two-component system, articulated by interactions between protein products of genes
and
, confers defense against heterotypic phage infection by PurpleHaze (cluster A/subcluster A3) or Alma (cluster A/subcluster A9) but is insufficient to confer defense against attack by the heterotypic phage Island3 (cluster I/subcluster I1). Therefore, based on heterotypic phage plating efficiencies on the Butters lysogen, additional prophage genes required for defense are implicated and further show specificity of prophage-encoded defense systems.
Many sequenced bacterial genomes, including those of pathogenic bacteria, contain prophages. Some prophages encode defense systems that protect their bacterial host against heterotypic viral attack. Understanding the mechanisms undergirding these defense systems is crucial to appreciate the scope of bacterial immunity against viral infections and will be critical for better implementation of phage therapy that would require evasion of these defenses. Furthermore, such knowledge of prophage-encoded defense mechanisms may be useful for developing novel genetic tools for engineering phage-resistant bacteria of industrial importance.
Duplicated ribosomal protein (Rp) gene families often encode highly similar or identical proteins with redundant or unique roles. Eukaryotic-specific paralogues RpL22e and RpL22e-like-PA are ...structurally divergent within the N terminus and differentially expressed, suggesting tissue-specific functions. We previously identified RpL22e-like-PA as a testis Rp. Strikingly, RpL22e is detected in immunoblots at its expected molecular mass (m) of 33 kD and at increasing m of ~43-55 kD, suggesting RpL22e post-translational modification (PTM). Numerous PTMs, including N-terminal SUMOylation, are predicted computationally. Based on S2 cell co-immunoprecipitations, bacterial-based SUMOylation assays and in vivo germline-specific RNAi depletion of SUMO, we conclude that RpL22e is a SUMO substrate. Testis-specific PTMs are evident, including a phosphorylated version of SUMOylated RpL22e identified by in vitro phosphatase experiments. In ribosomal profiles from S2 cells, only unconjugated RpL22e co-sediments with active ribosomes, supporting an extra-translational role for SUMOylated RpL22e. Ectopic expression of an RpL22e N-terminal deletion (lacking SUMO motifs) shows that truncated RpL22e co-sediments with polysomes, implying that RpL22e SUMOylation is dispensable for ribosome biogenesis and function. In mitotic germ cells, both paralogues localize within the cytoplasm and nucleolus. However, within meiotic cells, phase contrast microscopy and co-immunohistochemical analysis with nucleolar markers nucleostemin1 and fibrillarin reveals diffuse nucleoplasmic, but not nucleolar RpL22e localization that transitions to a punctate pattern as meiotic cells mature, suggesting an RpL22e role outside of translation. Germline-specific knockdown of SUMO shows that RpL22e nucleoplasmic distribution is sensitive to SUMO levels, as immunostaining becomes more dispersed. Overall, these data suggest distinct male germline roles for RpL22e and RpL22e-like-PA.
The proposed Drosophila melanogaster L23a ribosomal protein features a conserved C-terminal amino acid signature characteristic of other L23a family members and a unique N-terminal extension Koyama ...et al. (Poly(ADP-ribose) polymerase interacts with novel Drosophila ribosomal proteins, L22 and l23a, with unique histone-like amino-terminal extensions. Gene 1999; 226: 339-345), absent from Saccharomyces cerevisiae L25 that nearly doubles the size of fly L23a. The ability of fly L23a to replace the role of yeast L25 in ribosome biogenesis was determined by creating a yeast strain carrying an L25 chromosomal gene disruption and a plasmid-encoded FLAG-tagged L23a gene. Though affected by a reduced growth rate, the strain is dependent on fly L23a-FLAG function for survival and growth, demonstrating functional compatibility between the fly and yeast proteins. Pulse-chase experiments reveal a delay in rRNA processing kinetics, most notably at a late cleavage step that converts precursor 27S rRNA into mature 25S rRNA, likely contributing to the strain's slower growth pattern. Yet, given the essential requirement for L23(a)/L25 in ribosome biogenesis, there is a remarkable tolerance for accommodating the fly L23a N-terminal extension within the structure of the yeast ribosome. A search of available databases shows that the unique N-terminal extension is shared by multiple insect lineages. An evolutionary perspective on L23a structure and function within insect lineages is discussed.
The Taptic genome is the first to be annotated from the W cluster of mycobacteriophages infecting
mc
155. All 92 predicted open reading frames (ORFs) and a single tRNA specifying glycine (tRNA-gly) ...are transcribed rightward. Many functionally uncharacterized ORFs appear to be W cluster specific, as nucleotide similarity is shared only with other W cluster genomes.
Cluster BE1
bacteriophages belong to the
, with genome sizes over 130 kbp, and they contain direct terminal repeats of approximately 11 kbp. Eight newly isolated closely related cluster BE1 phages ...contain 43 to 48 tRNAs, one transfer-messenger RNA (tmRNA), and 216 to 236 predicted open reading frames (ORFs), but few of their genes are shared with other phages, including those infecting
species.
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