Dataset Information

RpL22e, but not RpL22e-like-PA, is SUMOylated and localizes to the nucleoplasm of Drosophila meiotic spermatocytes.

ABSTRACT: 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.

SUBMITTER: Kearse MG

PROVIDER: S-EPMC3720754 | biostudies-literature | 2013 May-Jun

REPOSITORIES: biostudies-literature

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RpL22e, but not RpL22e-like-PA, is SUMOylated and localizes to the nucleoplasm of Drosophila meiotic spermatocytes.

Kearse Michael G MG Ireland Jill A JA Prem Smrithi M SM Chen Alex S AS Ware Vassie C VC

Nucleus (Austin, Tex.) 20130501 3

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 RpL22 ...[more]

PMID: 23778934

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Project description:Previous data suggested that anastral spindles, morphologically similar to those found in oocytes, can assemble in a centrosome-independent manner in cells that contain centrosomes. It is assumed that the microtubules that build these acentrosomal spindles originate over the chromatin. However, the actual processes of centrosome-independent microtubule nucleation, polymerisation, and sorting have not been documented in centrosome-containing cells. We have identified two experimental conditions in which centrosomes are kept close to the plasma membrane, away from the nuclear region, throughout meiosis I in Drosophila spermatocytes. Time-lapse confocal microscopy of these cells labelled with fluorescent chimeras reveals centrosome-independent microtubule nucleation, growth, and sorting into a bipolar spindle array over the nuclear region, away from the asters. The onset of noncentrosomal microtubule nucleation is significantly delayed with respect to nuclear envelope breakdown and coincides with the end of chromosome condensation. It takes place in foci that are close to the membranes that ensheath the nuclear region, not over the condensed chromosomes. Metaphase plates are formed in these spindles, and, in a fraction of them, some degree of polewards chromosome segregation takes place. In these cells that contain both membrane-bound asters and an anastral spindle, the orientation of the cytokinesis furrow correlates with the position of the asters and is independent of the orientation of the spindle. We conclude that the fenestrated nuclear envelope may significantly contribute to the normal process of spindle assembly in Drosophila spermatocytes. We also conclude that the anastral spindles that we have observed are not likely to provide a robust back-up able to ensure successful cell division. We propose that these anastral microtubule arrays could be a constitutive component of wild-type spindles, normally masked by the abundance of centrosome-derived microtubules and revealed when asters are kept away. These observations are consistent with a model in which centrosomal and noncentrosomal microtubules contribute to the assembly and are required for the robustness of the cell division spindle in cells that contain centrosomes.

Dataset Information

RpL22e, but not RpL22e-like-PA, is SUMOylated and localizes to the nucleoplasm of Drosophila meiotic spermatocytes.

Publications

RpL22e, but not RpL22e-like-PA, is SUMOylated and localizes to the nucleoplasm of Drosophila meiotic spermatocytes.

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