Molecular & Biochemical Parasitology 193 (2014) 82–92 Contents lists available at ScienceDirect Molecular & Biochemical Parasitology Analysis of U3 snoRNA and small subunit processome components in the parasitic protist Entamoeba histolytica Ankita Srivastava a , Jamaluddin Ahamad a , Ashwini Kumar Ray a , Devinder Kaur 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 16 September 2013 Received in revised form 27 February 2014 Accepted 1 March 2014 Available online 12 March 2014 Keywords: rRNA processing Entamoeba histolytica Small subunit processome U3 snoRNA folding Pre-rRNA accumulation 5 ′ -ETS a b s t r a c t In the early branching parasitic protist Entamoeba histolytica, pre-rRNA synthesis continues when cells are subjected to growth stress, but processing slows down and unprocessed pre-rRNA accumulates. To gain insight into the regulatory mechanisms leading to accumulation, it is necessary to define the pre-rRNA processing machinery in E. histolytica. We searched the E. histolytica genome sequence for homologs of the SSU processome, which contains the U3snoRNA, and 72 proteins in yeast. We could identify 57 of the proteins with high confidence. Of the rest, 6 were absent in human, and 4 were non-essential in yeast. The remaining 5 were absent in other parasite genomes as well. Analysis of U3snoRNA showed that the E. histolytica U3snoRNA adopted the same conserved secondary structure as seen in yeast and human. The predicted structure was verified by chemical modification followed by primer extension (SHAPE). Further we showed that the predicted interactions of Eh U3snoRNA boxes A and A ′ with pre-18S rRNA were highly conserved both in position and sequence. The predicted interactions of 5 ′ -hinge and 3 ′ -hinge sequences of Eh U3 snoRNA with the 5 ′ -ETS sequences were conserved in position but not in sequence. Transcription of selected genes of SSU processome was tested by northern analysis, and transcripts of predicted sizes were obtained. During serum starvation, when unprocessed pre-RNA accumulated, the transcript levels of some of these genes declined. This is the first report on pre-rRNA processing machinery in E. histolytica, and shows that the components are well conserved with respect to yeast and human. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Ribosome biogenesis is a highly energy consuming process and is thus closely regulated with respect to cellular growth and nutri- ent availability [1,2]. It involves the synthesis of pre-rRNA which is co-transcriptionally processed and chemically modified into the mature rRNAs (18S, 5.8S and 25S/28S rRNAs). These, along with the separately transcribed 5S rRNA and ribosomal proteins assem- ble into the small- and large-ribosomal subunits [3]. Studies with model organisms e.g. yeast, Drosophila and mammals have pro- vided extensive information about the mechanism of pre-rRNA processing. The co-transcriptional initiation of this process has been elegantly visualized by electron microscopy, in the Miller ∗ Corresponding author at: School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110067, India. Tel.: +91 11 26704308. E-mail addresses: ankita26@gmail.com (A. Srivastava), j.ahamadjnu@gmail.com (J. Ahamad), ashwinires21@gmail.com (A.K. Ray), kaur.devbio@gmail.com (D. Kaur), alok.bhattacharya@gmail.com (A. Bhattacharya), sbjnu110@gmail.com, sb@mail.jnu.ac.in (S. Bhattacharya). chromatin spreads [4,5]. The pre-rRNA undergoes endonucleolytic cleavages at precise sites in the external and internal transcribed spacers (ETS and ITS respectively). These cleavages require the U3 snoRNA, which along with a large number of protein components forms a ribonucleoprotein complex called small subunit (SSU) pro- cessome [6,7]. U3 is a non-canonical C/D box-containing snoRNA that, unlike other snoRNAs which perform chemical modifications of rRNAs, has the specialized function of assisting in pre-rRNA processing and maturation [8]. It interacts with pre-rRNA by base pairing at specific sites within the 5 ′ -ETS and the pre-18S rRNA to carry out processing reactions at sites A0, A1 and A2 to release the mature 18S rRNA [9]. The secondary structure of U3 snoRNA consists of two major domains: a short 5 ′ -domain, and a longer 3 ′ -domain, con- nected by a hinge region [10,11]. The 5 ′ -domain, which contains the conserved sequences (GAC, box A ′ and box A), and the hinge region interact by complementary base-pairing with nucleotides at conserved locations in 18S rRNA and 5 ′ -ETS respectively [12,13]. The 3 ′ -domain is comprised of boxes C ′ , B, C and D, and is involved in interaction with proteins [14,15]. In addition to U3, other snoR- NAs are also involved in cleavage of pre-18S rRNA, although their http://dx.doi.org/10.1016/j.molbiopara.2014.03.001 0166-6851/© 2014 Elsevier B.V. All rights reserved.