Dataset Information

Identification of an evolutionarily divergent U11 small nuclear ribonucleoprotein particle in Drosophila.

ABSTRACT: Previous reports suggested that U11, in contrast to U12 or other small nuclear (sn)RNAs of the U12-type spliceosome, might be either highly divergent or absent in Drosophila melanogaster. Affinity purification of Drosophila U12-containing complexes has led to the identification of the fly U11 snRNA, which contains a potential U12-type 5' splice-site-interacting sequence, but whose sequence and length differs significantly from vertebrate and plant U11. Analysis of U12-type introns revealed an A-rich region directly downstream of Drosophila, but not human, U12-type 5' splice sites. This finding, coupled with the presence of a highly divergent U11 snRNA, and the apparent absence of Drosophila homologs of human U11 proteins, suggest that U12-type 5' splice site recognition might be different in flies. A comparison of U11 snRNAs that we have identified from vertebrates, plants, and insects, suggests that an evolutionarily divergent U11 snRNA may be unique to Drosophila and not characteristic of insects in general.

SUBMITTER: Schneider C

PROVIDER: S-EPMC470718 | biostudies-literature | 2004 Jun

REPOSITORIES: biostudies-literature

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Identification of an evolutionarily divergent U11 small nuclear ribonucleoprotein particle in Drosophila.

Schneider Claudia C Will Cindy L CL Brosius Jürgen J Frilander Mikko J MJ Lührmann Reinhard R

Proceedings of the National Academy of Sciences of the United States of America 20040621 26

Previous reports suggested that U11, in contrast to U12 or other small nuclear (sn)RNAs of the U12-type spliceosome, might be either highly divergent or absent in Drosophila melanogaster. Affinity purification of Drosophila U12-containing complexes has led to the identification of the fly U11 snRNA, which contains a potential U12-type 5' splice-site-interacting sequence, but whose sequence and length differs significantly from vertebrate and plant U11. Analysis of U12-type introns revealed an A- ...[more]

PMID: 15210936

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Project description:Twenty years since the publication of the Plasmodium falciparum and P. berghei genomes one-third of their protein-coding genes still lack functional annotation. In the absence of sequence and structural homology, protein-protein interactions can facilitate functional prediction of such orphan genes by mapping protein complexes in their natural cellular environment. The Plasmodium nuclear pore complex (NPC) is a case in point: it remains poorly defined; its constituents lack conservation with the 30+ proteins described in the NPC of many opisthokonts, a clade of eukaryotes that includes fungi and animals, but not Plasmodium. Here, we developed a labeling methodology based on TurboID fusion proteins, which allows visualization of the P. berghei NPC and facilitates the identification of its components. Following affinity purification and mass spectrometry, we identified 4 known nucleoporins (Nups) (138, 205, 221, and the bait 313), and verify interaction with the putative phenylalanine-glycine (FG) Nup637; we assigned 5 proteins lacking annotation (and therefore meaningful homology with proteins outside the genus) to the NPC, which is confirmed by green fluorescent protein (GFP) tagging. Based on gene deletion attempts, all new Nups - Nup176, 269, 335, 390, and 434 - are essential to parasite survival. They lack primary sequence homology with proteins outside the Plasmodium genus; albeit 2 incorporate short domains with structural homology to human Nup155 and yeast Nup157, and the condensin SMC (Structural Maintenance Of Chromosomes 4). The protocols developed here showcase the power of proximity labeling for elucidating protein complex composition and annotation of taxonomically restricted genes in Plasmodium. It opens the door to exploring the function of the Plasmodium NPC and understanding its evolutionary position. IMPORTANCE The nuclear pore complex (NPC) is a platform for constant evolution and has been used to study the evolutionary patterns of early-branching eukaryotes. The Plasmodium NPC is poorly defined due to its evolutionary divergent nature making it impossible to characterize it via homology searches. Although 2 decades have passed since the publication of the Plasmodium genome, 30% of the genes still lack functional annotation. Our study demonstrates the ability of proximity labeling using TurboID to assign function to orphan proteins in the malaria parasite. We have identified a total of 10 Nups that will allow further study of NPC dynamics, structural elements, involvement in nucleocytoplasmic transport, and unique non-transport functions of nucleoporins that provide adaptability to this malaria parasite.

Dataset Information

Identification of an evolutionarily divergent U11 small nuclear ribonucleoprotein particle in Drosophila.

Publications

Identification of an evolutionarily divergent U11 small nuclear ribonucleoprotein particle in Drosophila.

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