Transcriptomic analysis of ribosome biogenesis and pre-rRNA processing during growth stress in Entamoeba histolytica.

Ribosome biogenesis, a multi-step process involving transcription, modification, folding and processing of rRNA, is the major consumer of cellular energy. It involves sequential assembly of ribosomal proteins (RP)s via more than 200 ribogenesis factors. Unlike model organisms where transcription of rRNA and RP genes slows down during stress, in Entamoeba histolytica, pre-rRNA synthesis continues, and unprocessed pre-rRNA accumulates. Northern hybridization from different spacer regions depicted the accumulation of unprocessed intermediates during stress. To gain insight into the vast repertoire of ribosome biogenesis factors and understand the major components playing role during stress we computationally identified ribosome biogenesis factors in E. histolytica. Of the ∼279 Saccharomyces cerevisiae proteins, we could only find 188 proteins in E. histolytica. Some of the proteins missing in E. histolytica were also missing in humans. A number of proteins represented by multiple genes in S. cerevisiae had a single copy in E. histolytica. Interestingly E. histolytica lacked mitochondrial ribosome biogenesis factors and had far less RNase components compared to S. cerevisiae. Transcriptomic studies revealed the differential regulation of ribosomal factors both in serum starved and RRP6 down-regulation conditions. These included the NEP1 and TSR3 proteins that chemically modify 18S-rRNA. Pre-rRNA precursors accumulate upon downregulation of the latter proteins in S. cerevisiae and humans. These data reveal the major factors that regulate pre-rRNA processing during stress in E. histolytica and provide the first complete repertoire of ribosome biogenesis factors in this early-branching protist.

Stress-induced nuclear depletion of Entamoeba histolytica 3'-5' exoribonuclease EhRrp6 and its role in growth and erythrophagocytosis.

The 3'-5' exoribonuclease Rrp6 is a key enzyme in RNA homeostasis involved in processing and degradation of many stable RNA precursors, aberrant transcripts, and noncoding RNAs. We previously have shown that in the protozoan parasite Entamoeba histolytica, the 5'-external transcribed spacer fragment of pre-rRNA accumulates under serum starvation-induced growth stress. This fragment is a known target of degradation by Rrp6. Here, we computationally and biochemically characterized EhRrp6 and found that it contains the catalytically important EXO and HRDC domains and exhibits exoribonuclease activity with both unstructured and structured RNA substrates, which required the conserved DEDD-Y catalytic-site residues. It lacked the N-terminal PMC2NT domain for binding of the cofactor Rrp47, but could functionally complement the growth defect of a yeast rrp6 mutant. Of note, no Rrp47 homologue was detected in E. histolytica Immunolocalization studies revealed that EhRrp6 is present both in the nucleus and cytosol of normal E. histolytica cells. However, growth stress induced its complete loss from the nuclei, reversed by proteasome inhibitors. EhRrp6-depleted E. histolytica cells were severely growth restricted, and EhRrp6 overexpression protected the cells against stress, suggesting that EhRrp6 functions as a stress sensor. Importantly EhRrp6 depletion reduced erythrophagocytosis, an important virulence determinant of E. histolytica This reduction was due to a specific decrease in transcript levels of some phagocytosis-related genes (Ehcabp3 and Ehrho1), whereas expression of other genes (Ehcabp1, Ehcabp6, Ehc2pk, and Eharp2/3) was unaffected. This is the first report of the role of Rrp6 in cell growth and stress responses in a protozoan parasite.

Transcriptomic analysis reveals novel downstream regulatory motifs and highly transcribed virulence factor genes of Entamoeba histolytica.

BACKGROUND: Promoter motifs in Entamoeba histolytica were earlier analysed using microarray data with lower dynamic range of gene expression. Additionally, previous transcriptomic studies did not provide information on the nature of highly transcribed genes, and downstream promoter motifs important for gene expression. To address these issues we generated RNA-Seq data and identified the high and low expressing genes, especially with respect to virulence potential. We analysed sequences both upstream and downstream of start site for important motifs. RESULTS: We used RNA-Seq data to classify genes according to expression levels, which ranged six orders of magnitude. Data were validated by reporter gene expression. Virulence-related genes (except AIG1) were amongst the highly expressed, while some kinases and BspA family genes were poorly expressed. We looked for conserved motifs in sequences upstream and downstream of the initiation codon. Following enrichment by AME we found seven motifs significantly enriched in high expression- and three in low expression-classes. Two of these motifs (M4 and M6) were located downstream of AUG, were exclusively enriched in high expression class, and were mostly found in ribosomal protein, and translation-related genes. Motif deletion resulted in drastic down regulation of reporter gene expression, showing functional relevance. Distribution of core promoter motifs (TATA, GAAC, and Inr) in all genes revealed that genes with downstream motifs were not preferentially associated with TATA-less promoters. We looked at gene expression changes in cells subjected to growth stress by serum starvation, and experimentally validated the data. Genes showing maximum up regulation belonged to the low or medium expression class, and included genes in signalling pathways, lipid metabolism, DNA repair, Myb transcription factors, BspA, and heat shock. Genes showing maximum down regulation belonged to the high or medium expression class. They included genes for signalling factors, actin, Ariel family, and ribosome biogenesis factors. CONCLUSION: Our analysis has added important new information about the E. histolytica transcriptome. We report for the first time two downstream motifs required for gene expression, which could be used for over expression of E. histolytica genes. Most of the virulence-related genes in this parasite are highly expressed in culture.

Maxillofacial trauma among Indians.

Orofacial injuries constitute the medico-legal cases reported, especially, in cases associated with road traffic accidents, assaults, and violence making it an emerging healthcare problem. Therefore, it is of interest to document data on the maxillofacial trauma and fractures among Indians. 150 subjects within the age of 15 to 60 years with maxillofacial fractures, detailed medical history including demographics, radiographs, medical history, associated injuries, and etiology of fractures were used for this study. Sites for both maxillary and mandibular fractures were noted. The type of intubation (medical insertion procedure) used and post-operative complications were also recorded. Lefort I, II, and III fractures were seen in 4%, 12%, 6% subjects respectively, whereas, ZMC fracture was seen in 66% study subjects. Mandibular fractures were most commonly seen in the para-symphysis region with 30% subjects followed by condylar region with 28.66% subjects. Data shows that maxillofacial trauma has a high incidence in India with RTA (road traffic accidents being the most common reason for the trauma seen in young males with significant concomitant injuries. Most common fracture is seen in mandible region. However, they can be managed well with very few postoperative complications.