Nuclear dimorphism is a characteristic feature of the ciliated protists. The macronucleus (MAC) is transcriptionally active during vegetative growth, whereas the germline micronucleus (MIC) is completely transcriptionally silent during vegetative growth. However, the active transcription of the MIC genome is essential for sexual reproduction.
During the sexual reproduction of ciliated protist Tetrahymena, its MIC undergoes meiosis, at the same time, the RNA polymerase II (RNAPII) is recruited to the MIC, and then double stranded non-coding RNAs are generated by bi-directional transcription of the germline genome. The double stranded non-coding RNAs are further processed into small RNAs (called scnRNAs) by Dicer homolog Dcl1, and are guiding the deletion of one-third of the genome from the developing progeny MAC. Although the molecular mechanisms underlying the small RNA-directed DNA elimination event during the sexual process of conjugation have been intensively studied, the activation and regulation of germline transcription is still a mystery. By deleting genes that are specifically expressed in meiotic prophase of Tetrahymena, we have identified three (RIB1, MIP1, MIP2) genes that are involved in regulation of germline transcription. Knockout of any of them blocks the accumulation of non-coding RNAs from the germline nucleus and prevents the DNA elimination process, indicating that these three genes are essential for germline transcription. Rib1 and Mip1 proteins are specifically localized in the MIC during meiosis. Interestingly, the localization of Rib1 is similar to that of the RNAPII specific subunit Rpb3 in the meiotic prophase MIC. However, the localization of Rpb3 is not affected by the deletion of RIB1, indicating that Rib1 might be critical for activating RNAPII transcription but not required for the localization of RNAPII. The deletion of MIP1 disrupted the localization of Rib1 and Rpb3 to chromatin, suggesting that Mip1 plays a role in the recruitment of RNAPII and possibly its auxiliary factors, such as Rib1, to the MIC. Also in a mip2 deletion, Rib1 and Rpb3 are mislocalized. Therefore, the Mip2 protein is possibly required for micronuclear import of Rib1 and the proper distribution of RNAPII within the MIC.
Finally, we noticed that the knockout of MIP1 and MIP2 lead to the extension of meiotic prophase, which might suggest delayed repair of meiotic DNA double-strand breaks in mip1Δ and mip2Δ cells.