**Analysis of 3'UTR of** *Prnp* **Gene in Mammals: Possible Role of Target Sequences of miRNA for TSE Sensitivity in Bovidae and Cervidae**

Daniel Petit, Jean-Michel Petit and François Gallet *UGMA, UMR 1061 INRA/University of Limoges France*

### **1. Introduction**

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Transmissible spongiform encephalopathies (TSEs) or prion diseases are neurodegenerative diseases with an inexorably fatal outcome. They affect both human and other mammals, and are especially frequently found in Bovidae and Cervidae families (Aguzzi & Sigurdson, 2004). In the first family, sheep and goats can spontaneously develop the prion disease or scrapie (Grenn et al., 2008), whereas cattle only develop a disease, known as Bovine Spongiform Encephalopathy (BSE), after a contact with the infectious prion protein (Ducrot et al., 2008). In the second family, the chronic wasting disease (CWD) has been described in mule deer and elk. Besides these two Ruminant families, TSE was also retrieved in Carnivora, as Felidae (feline spongiform encephalopathy) and Mustelidae (transmissible mink encephalopathy) (Miller et al., 2008; Sigurdson & Miller, 2003). All these diseases are characterized by the accumulation of PrPSc, the abnormally folded isoform of the cellular prion protein (PrPC) in the brain (Prusiner 1998; Norrby 2011).

The cellular prion protein is a glycosylphosphatidylinositol anchored glycoprotein of 256 amino acids in sheep (Bovidae, subfamily Caprinae) and Cervidae. The N-ter of the protein is mainly unstructured while the C-terminal domain is globular. The C-ter domain is highly structured and is stabilized by an intramolecular disulfide bound. It contains three -helices and a short -sheet. TSE is a conformational disease, where change from PrPC to PrPSc involves an increase in -sheet content from 3% to 40%, and a decrease in -helical structure from 40% to 30% that modify irreversibly the protein folding (Cohen et al., 1998; Smirnovas et al., 2009).

In a previous work, we showed that the ARQ/ARQ genotype is rather sensitive to the TSE in sheep and goat but protective in pig and rabbit, known as resistant species [Martin et al., 2009]. Otherwise, several studies point out that the expression level of the prion gene could modulate the onset of TSE, particularly in cattle (Sander et al., 2004-2006; Haase et al., 2007). The involvement of the 5' promoter region has been investigated in sheep (Saunders et al., 2009) and cattle (Xue et al., 2008) and it appears that polymorphism in this region could induce different responses to scrapie (Marcos-Carcavilla et al., 2008) and BSE (Brunelle et al., 2008). The analysis of a 23 pb insertion/deletion polymorphism in German and Swiss breed cattle revealed that the deletion is associated to a higher expression level, more frequently

Analysis of 3'UTR of Prnp Gene in Mammals:

Table 1. List of mammalian studied species

**3.1 Position of insertions and deletions** 

**3. Results** 

Griffiths-Jones (2011).

Possible Role of Target Sequences of miRNA for TSE Sensitivity in Bovidae and Cervidae 525

sequences of mi-RNAs were detected with mirBase (www.mirbase.org/), following the papers of Griffiths-Jones (2004), Griffiths-Jones et al. (2006 and 2008), and Kozomara &

Species names Access numbers in NCBI

*Bos taurus* NM\_181015 *Canis familiaris* NC\_006606.2 *Cervus elaphus* EU032284

*Equus caballus* AAWR02026367.1 *Felis catus* ACBE01091825.1 *Homo sapiens* NC\_000020.10, U29185 *Lama pacos* ABRR01260217 *Loxodonta africana* AAGU03041236.1 *Macropus eugenii* ABQO010016931.1 *Monodelphis domestica* AAFR03004045.1 *Mus musculus* NM\_011170, U29186 *Mustella putorius* AEYP01079589.1 *Myodes glareolus* EF455012

*Myotis lucifugus* AAPE02032418.1 *Ochotona princeps* AAYZ01152834.1 *Odocoileus hemionus* AY330343 *Oryctolagus cuniculus* NW\_003159242.1 *Ovis aries* NM\_00100948.1, U67922 *Procavia capensis* ABRQ01131540.1 *Pteropus vampyrus* ABRP01225173.1 *Rattus norvegicus* NC\_005102.2 *Saimiri sciureus* ti|1176383476 *Sorex araneus* AALT01429291.1 *Spermophilus tridecemlineatus* AAQQ01175405.1 *Sus scrofa* NM\_001008687 *Tarsius syrichta* ABRT010290102.1 *Tursiops truncatus* ABRN01336586.1

In order to visualise the most conservative sites of the 3'UTR, we used the method developed in Petit et al. (2006) and Martin et al. (2009). Briefly, the alignment of sequences, cleaned from insertions, was treated by Parsimony program of Phylip package vers. 3.69 (Felsenstein, 2004), to export the number of changes site by site. For each site, this number was divided by the number of sequences, allowing drawing the profile of site change rates.

The length of the 3'UTR region is highly variable as the murine sequence comprises about 1250 pb whereas the bovine one is more than 3500 pb long and possesses two potential polyadenylation signals that are separated by about 1300 bp. Such a result is also observed in sheep and mule deer. The difference is mainly explained by a series of transposable

found in cattle affected with classical BSE. Moreover, in vitro studies showed that the possibility to infect neurosphere cultures with scrapie prion is linked to an over-expression of PrPC (Giri et al., 2006). Although the determinism of prion disease is multifactorial (prion strain and prion protein sequence, see Doherr, 2003), it seems that a high expression of PrPC gene is necessary (Krejciova et al., 2011).

In numerous organisms, post-transcriptional gene regulation involves small (around 18-25 nucleotides long) non-coding RNA molecules, the microRNAs (miRNAs). They recognise specific target sequences in the 3'UTR of some transcripts, mediating their silencing (Fabian et al., 2010) by inducing their degradation or by inhibiting their translation. According to the accuracy of the complementarity between both sequences, each miRNA can regulate up to hundreds of genes. Using microarrays, Saba et al. (2008) evidenced 15 miRNAs, potentially controlling the transcript amount of more than 100 genes. These micro-RNAs are deregulated in the brain of scrapie affected mouse.

In their comparative analysis of *Prnp* organization in human, mouse and sheep, Lee et al. (1998) showed the high conservation of the 3'-UTR regions and suggested their role in mRNA stability. Moreover, they evidenced insertions of transposable elements in the sheep gene. In their more recent work, Premzl and Gamulin (2007) could easily align the part close to the poly-adenylation signal in a wide range of mammals. Taken together, these data suggest that alignments of mammalian 3'UTRs contain reliable information. The regulatory sequences borne by the 3'-UTR are involved in mRNA processing, transport, stability, and translation. As the 3'-UTRs harbor recognition sites for diverse RNA-binding proteins that regulate gene expression as well as active microRNA target sites, our strategy was to compare the 3'UTR of the *Prnp* gene, from the sequence terminus codon to the polyadenylation signal in different Mammal lineages. We expect to find in the most frequently families affected by TSE, i.e., Bovidae and Cervidae, oddities in their potential miRNA targets. Three types of results are predicted to be obtained. In the first type, based on the miRNA-related gene silencing, we predict that Bovidae and Cervidae families lack some targets widely present in other Mammals. In the second type, we predict that target sequences are present in DNA from most mammalian lineages, including the both critical families, but a loss of corresponding miRNA may occur. The last type, although more unlikely, would be to detect targets only harboured by Bovidae and Cervidae, which would lead to the activation of gene expression, as shown by Vasudevan et al. (2007) in a model of cellular stress. In brief, we search for potential targets that could affect repression mechanisms of *Prnp* gene expression, and thus enhance the sensitivity of Bovidae and Cervidae to the disease.
