**4. Discussion**

530 A Bird's-Eye View of Veterinary Medicine

common to *E. caballus*, *F. catus*, *L. pacos* and *S. scrofa*, and Mir-15B/369 to *E. caballus*, *H.* 

Fig. 6. Position of putative targets within the large deletion common to Bovidae-Cervidae.

Fig. 7. Alignment of overlapping target sequences in the region of the large deletion

common to Bovidae-Cervidae. Green background: conservation in ≥ 75 % sequences; yellow

Bovidae and Cervidae are the only species in which transposable elements are present in the transcript of *Prnp.* The poly-adenylation site common with other mammals is situated downstream from Mariner element. However, there is an extra potential poly-adenylation site, downstream to the SINE/BovA. Few targets for Mirs are present in these regions and

The grey box corresponds to the target present in human.

background: conservation in ≥ 50 % sequences.

common to *B. taurus*, *O. aries* and *O. hemionus* (Table 4).

*sapiens*, *F. catus*, and *S. scrofa*.

The most interesting feature in the comparative analysis of 3'UTR region of mammalian *Prnp* genes is the occurrence of 2 events common to Bovidae and Cervidae, i.e., a 177 pblong deletion and 3 insertions of transposable elements. These insertions were previously described in sheep by Lee et al. (1998), but not in Cervidae. To simplify the discussion, the large deletion common to Bovidae-Cervidae will be abbreviated as LDCBC in the following. The mammalian distribution of insertions and LDCBC fit with the phylogeny within Artiodactyla, since Bovidae and Cervidae were shown to be sister families by Kuznetsova et al. (2005) from 12S and 16S rRNA analyses. In contrast, the more basally genera *Lama* (Camelidae), *Sus* (Suidae) and *Tursiops* (Delphinidae) within Cetartiodactyla do not possess the LDCBC and the 3 insertions. Unfortunately, none studied species harboured either the LDCBC or the 3 insertions. The sequencing of *Giraffa camelopardalis* could bring an

Analysis of 3'UTR of Prnp Gene in Mammals:

many approaches still need to be explored such as miRNAs.

and Cervidae could be explained by the lack of several key targets.

We thank the Région Limousin for financial support.

Nat. Rev. Immunol., 4(9), 725-736.

species known as resistant..

**5. Conclusion** 

**6. Acknowledgments** 

**7. References** 

285.

Research, 4, 36-43.

15524-15529.

Possible Role of Target Sequences of miRNA for TSE Sensitivity in Bovidae and Cervidae 533

The sensitivity to TSE has certainly multiple origins according to mammals. For example, the 3'UTR sequence of *M. putorius*, known as sensitive (Miller et al., 2008), presents no indel in the first 600 pb. Interestingly, this species as well as *Myodes glareolus*, another sensitive species (Di Bari et al., 2008), lack both targets for Mir 4763 and 15B/369, in contrast to the

The sensitivity of a given Mammal to TSE depends on many factors, as shown by the network analyses conducted during the development of the disease. But even if many genes have been proved to be involved, the expression level of *Prnp* gene plays a crucial role and

The 3'-UTR sequence of *Prnp* gene is well conserved with Eutherians, and especially in the first 600 pb downstream to stop codon, suggesting a role in regulation of stability of the mRNA. In the 3'-UTR part of *SPRN*, a gene close to *Prnp*, Premzl & Gamulin (2007) showed a target for Mir-34a shared by several orders of mammals. It is unlikely that such a unique target is present in the case of mammalian *Prnp*. Our analyses rather suggest an interaction between several targets which could modulate the stability of this transcript. The combination of acting Mirs could vary among mammals and the high sensitivity of Bovidae

Aguzzi, A & Sigurdson, C (2004). Antiprion immunotherapy: to suppress or to stimulate?

Bottoni, A, Piccin, D, Tagliati, F, Luchin, A, Zatelli, MC, & degli Uberti, EC. (2005). miR-15a

Brunelle, BW, Greenlee, JJ, Seabury, CM, Brown, CE 2nd, & Nicholson, EM.

Calin, GA, Dumitru, CD, Shimizu, M, Bichi, R, Zupo, S, Noch, E, Aldler, H, Rattan, S,

Chekulaeva, M, & Filipowicz, W. (2009) Mechanisms of miRNA-mediated posttranscriptional regulation in animal cells. Curr. Opin. Cell Biol., 21, 452–60. Chung, GE, Yoon, JH, Myung, SJ, Lee, JH, Lee, SH, Lee, SM, Kim, SJ, Hwang, SY, Lee, HS, &

and miR-16-1 down-regulation in pituitary adenomas. J. Cell Physiol., 204(1), 280-

(2008). Frequencies of polymorphisms associated with BSE resistance differ significantly between Bos taurus, Bos indicus, and composite cattle. BMC Vet.

Keating, M, Rai, K, Rassenti, L, Kipps, T, Negrini, M, Bullrich, F, & Croce, CM. (2002). Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci U.S.A., 99(24),

Kim, CY. (2010). High expression of microRNA-15b predicts a low risk of tumor

interesting issue, given its intermediary position between Bovidae-Cervidae group and the Camelidae. Consequently, it is not possible to assign a specific role of the LDCBC and or of the 3 insertions to the stability of *Prnp* RNA. Their potential influence will be assessed independently.

As explained in introduction, several types of potential target sites of micro-RNAs are to be explored according to their position relatively to the LDCBC: (i) between the stop codon and the LDCBC (all Mammals), (ii) in the sequence corresponding to the LDCBC (all Mammals except Bovidae-Cervidae), and (iii) in the inserted elements particular to Bovidae-Cervidae. Generally, miRNAs inhibit protein synthesis either by the repression of the translation and/or by the activation of the deadenylation leading to the degradation of the targeting mRNA (Eulalio et al., 2008; Chekulaeva & Filipowicz, 2009). Multiples sites, either for the same or different miRNAs, are usually required for a more effective repression, and they tend to act cooperatively when they are close to each other (Doench et al., 2004; Grimson et al., 2007). As most microRNA play a role in repression, a special attention will be brought to targets present in most mammals but absent in Bovidae-Cervidae. However, targets restricted to Bovidae-Cervidae could have significance through an activating role in stability of transcript (Vasudevan et al., 2007).

In the sequence comprised between stop codon and LDCBC, the most spread target within mammals corresponds the group Mir-3918/509-519. According to fig. 3, this target is situated in one of the most conservative regions in 3'UTR of PRNP gene. Its possible involvement could be interpreted as the result of an under-expression of the corresponding Mirs in Bovidae-Cervidae, according to the second type mentioned in introduction section. It would be interesting to test experimentally this idea.

In the sequence corresponding to the large deletion common to Bovidae-Cervidae, two targets are interesting to consider, corresponding to Mir4763 and Mir-15B/369. According to literature, Mir15b belong to a small family of Mirs comprising also Mir15a, Mir16a, Mir16b, and Mir195 in Mammals. Interestingly, Mir15b as well as Mir15a have proved to be involved as repressing cancers in human. It has been shown an association between deletion of Mir15a gene, together with Mir16 in the same cluster in HSA chromosome 13q14, and pituitary tumors (Bottoni et al., 2005). The same authors showed that the expression level of these 2 Mirs is inversely correlated to the pituitary adenoma growth. Another study of Calin et al., 2002 showed that deletions in the same cluster of Mir 15b-Mir16 are associated to chronic lymphocytic leukemia. Similarly, a high expression level of Mir15b is linked to a low proliferation of hepatocellular carcinoma (Chung et al., 2010). As for Mir369, Williams et al. (2007) showed that members of family Mir-154 are especially expressed in mouse and human foetal, but not adult, lung.

The presence of transposable elements in the *Prnp* transcript of Bovidae and Cervidae is an interesting feature. It is unlikely that they contribute to the stability of mRNA, as no publications support this view. However, these transposable elements contain putative targets for Mirs that confer new possibility of regulation, as shown in mammals (Smalheiser & Torvik, 2005). These putative Mirs could play a role in the stability of *Prnp* gene. We could bring the same hypothesis to the targets of Mir-569/155, 376, 15b, present in the region proximal to stop codon.

The sensitivity to TSE has certainly multiple origins according to mammals. For example, the 3'UTR sequence of *M. putorius*, known as sensitive (Miller et al., 2008), presents no indel in the first 600 pb. Interestingly, this species as well as *Myodes glareolus*, another sensitive species (Di Bari et al., 2008), lack both targets for Mir 4763 and 15B/369, in contrast to the species known as resistant..

The sensitivity of a given Mammal to TSE depends on many factors, as shown by the network analyses conducted during the development of the disease. But even if many genes have been proved to be involved, the expression level of *Prnp* gene plays a crucial role and many approaches still need to be explored such as miRNAs.
