Molecular characterization of telomeric noncoding RNAs in Leishmania major

Author(s)

Edna Gicela Ortiz Morea

• Document type: Doctoral Thesis
• Press: Botucatu. 2018-05-24.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências. Botucatu
• Defense date: 2018-03-23
• Advisor: Maria Isabel Nogueira Cano

Abstract

Protozoan from Leishmania genus are the etiologic agents of leishmaniasis, a tropical disease that presents different clinical manifestations and affect million people in Brazil and in the world. There are no eficiente vacines nor treatment protocols against leishmaniasis, therefore, it is crucial to improve the knowledge about Leishmania molecular biology and physiology for the development of new therapies. Actually, telomeres have been considered potential molecular targets as they are responsible to maintain genome stability. Leishmania telomeres similarly to other eukaryotes, are nucleoprotein structures found at the end of the chromosomes maintained by telomerase. They can be regulated by different mechanisms such as the action of long non-coding telomeric RNAs (lncRNAs). Among the telomeric lncRNAs are the telomerase RNAcomponent (TER) which is crucial for telomerase activity because it contains the template sequence that is copied by telomerase during telomere elongation. TER was recently identified in Leishmania although there is no information about its function and biogenesis.TERRA, the Telomere Repeat containing RNA is other telomeric RNA that it is involved with telomere length regulation telomere damage response and alterations in the composition of telomeric chromatin and is essential for telomere maintenance. In this work, we identified TERRA transcripts in the three Leishmania developmental stages (amastigote, promastigote e metacyclic). We interrogate three independent SL-Seq libraries obtained from total RNA obtained from Leishmania developmental stages and search for transcripts that present the sequence leader (SL) signal located upstream the subtelomeric/telomeric repeats as a mark for mRNA maturation. In addition, we had also search the L. major genome for the presence of Leishmania specific subtelomeric elements, the Conserved Sequence Boxes (CSB) and putative subtelomeric CpG islands. Using this approach, we found 7 different types of chromosome ends in L. major. In silico results were validated using Northern blot and RT-PCR/RT-qPCR. We confirmed the existence of TERRA transcripts of variable length and size, which also present different expression profiles depending on the parasite life stage. Our analyses also revealed the existence of polyadenilated TERRA transcripts from some but not all chromosome ends. In addition, telomere length analysis was done using parasites originated from a single developmental cycle and from promastigote and metacyclic forms maintained in vitro for up to 6 successive replicas in culture. The results showed that during a single developmental cycle amastigote forms present shorter telomeres than promastigotes and metacyclic derived there from. In addition telomeres length progressively shorten in promastigote and derived metacyclic during successive in vitro cultivation, or after increasing population doubling. We also observed that parasites showing progressive telomeres shortening have increased levels of TERRA transcripts. Therefore, these results suggest that i) TERRA expression is probably regulated at the transcriptional level during L. major development, ii) TERRA may be directly involved in parasite telomere maintenance, probably acting as a negative regulator of telomerase activity, and iii) probably promastigotes and derived metacyclic forms enter into replicative senescence after several population derived therefrom enter into replicative senescence after several population doubling cycles in vitro. To identified putatives CpG islands at subtelomeric position, we first performed digestion of DNA with methylation sensitive restriction enzymes. Although these results suggested the existence of methylated DNA at the subtelomeric putative CpG islands, bisulfite treatment followed by DNA sequencing did not confirm these findings. We had also initiate functional studies about a L. major TER component. We cloned, sequenced and subcloned TER gene (leishTER) in a Leishmania expression vector. In addition, we also design a CRISPR/Cas9 system to genetic edit the L. major TER in the parasite genome. And finally, we have standardized the use of antisense morpholine oligonucleotides as a tool to inhibit the maturation of the Leishmania TER component. This later approache will be valuable to study the functions of genes encoding protein and lncRNA in these protozoa, since only a few Leishmania species present the RNA interference machinery. (AU)

• FAPESP's process: 16/06883-0 - Identification and characterization of noncoding RNAs of the type TERRA and/or its counterparts expressed from subtelomeric region in Leishmania major
• Grantee: Edna Gicela Ortiz Morea
• Support Opportunities: Scholarships in Brazil - Doctorate