Molecular & Biochemical Parasitology 192 (2013) 30–38 Contents lists available at ScienceDirect Molecular & Biochemical Parasitology The ribosomal RNA transcription unit of Entamoeba invadens: Accumulation of unprocessed pre-rRNA and a long non coding RNA during encystation Sandeep Ojha a , Nishant Singh a , Alok Bhattacharya b , Sudha Bhattacharya a,∗ a School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110067, India b School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India a r t i c l e i n f o Article history: Received 18 September 2013 Received in revised form 25 October 2013 Accepted 29 October 2013 Available online 5 November 2013 Keywords: Circular rDNA Long non coding RNA Entamoeba invadens Intergenic spacer transcript Pre-rRNA accumulation a b s t r a c t The ribosomal RNA genes in Entamoeba spp. are located on extrachromosomal circular molecules. Unlike model organisms where rRNA transcription stops during growth stress, Entamoeba histolytica continues transcription; but unprocessed pre-rRNA accumulates during stress, along with a novel class of circular transcripts from the 5  -external transcribed spacer (ETS). To determine the fate of rRNA transcription during stage conversion between trophozoite to cyst we analyzed Entamoeba invadens, a model system for differentiation studies in Entamoeba. We characterized the complete rDNA transcription unit by mapping the ends of pre-rRNA and mature rRNAs. The 3  end of mature 28S rRNA was located 321 nt downstream of the end predicted by sequence homology with E. histolytica. The major processing sites were mapped in external and internal transcribed spacers. The promoter located within 146 nt upstream of 5  ETS was used to transcribe the pre-rRNA. On the other hand, a second promoter located at the 3  end of 28S rDNA was used to transcribe almost the entire intergenic spacer into a long non coding (nc) RNA (>10 kb). Interestingly we found that the levels of pre-rRNA and long ncRNA, measured by northern hybridization, decreased initially in cells shifted to encystation medium, after which they began to increase and reached high levels by 72 h when mature cysts were formed. Unlike E. histolytica, no circular transcripts were found in E. invadens. E. histolytica and E. invadens express fundamentally different ncRNAs from the rDNA locus, which may reflect their adaptation to different hosts (human and reptiles, respectively). This is the first description of rDNA organization and transcription in E. invadens, and provides the framework for further studies on regulation of rRNA synthesis during cyst formation. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Ribosomal RNA genes are typically highly repetitive, and are organized as tandem repeats on one or more chromosomes. Each rRNA gene contains an rRNA transcription unit which is transcribed into a polycistronic pre-rRNA. This undergoes specific processing to yield the mature rRNA species (18S, 5.8S and 28S rRNAs), while the external- and internal transcribed spacers (ETS and ITS respectively) are excised and degraded [1–3]. Consecutive rRNA transcription units are separated by intergenic spacers (IGS) which contain important transcriptional regulatory sequences, like pro- moters, enhancers and terminators [4,5]. Although rRNA coding sequences are amongst the most highly conserved in evolution, the IGS length and sequence is very divergent even in closely related ∗ Corresponding author. Tel.: +91 11 26704308; fax: +91 11 26741502. E-mail addresses: sandy.mbt@gmail.com (S. Ojha), nishant900@gmail.com (N. Singh), alok.bhattacharya@gmail.com (A. Bhattacharya), sbjnu110@gmail.com, sb@mail.jnu.ac.in (S. Bhattacharya). species. It was earlier thought that these spacers are not tran- scribed, but studies in a number of model systems like Xenopus [6], Drosophila [7] mammals and yeast [8,9] have shown the pres- ence of IGS transcripts, although in much less abundance than the rRNAs. Whereas the IGS in yeast is transcribed by RNA Pol II [10], those in Xenopus [11,12], Drosophila [13] and mouse rDNA are tran- scribed by RNA Pol I. Additionally, only a small part of the ∼30 kb IGS in mouse is transcribed to give a ∼2 kb RNA [14]. On the other hand in Xenopus and Drosophila almost the entire IGS is transcribed, save a ∼200 nt stretch upstream of the pre-rRNA transcription start point [6,13]. More recently the regulatory roles of IGS transcripts have been demonstrated. In mouse these transcripts are directly involved in maintaining the epigenetic silencing of a subset of rRNA genes [15]. In yeast the IGS transcripts are involved in recombina- tion within the rDNA array and in rDNA copy number control [9]. Thus it appears that the non coding (nc) RNAs encoded by the IGS have diverse origins and may have evolved a variety of species- specific functions. The transcription and processing of rRNA is tightly regulated in response to cell division, differentiation and environmental 0166-6851/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.molbiopara.2013.10.002