Ribozymes of the hepatitis delta virus: Recent findings on their structure, mechanism of catalysis and possible applications.

Although the delta ribozymes have been studied for more than ten years the most important information concerning their structure and mechanism of catalysis were only obtained very recently. The crystal structure of the genomic delta ribozyme turns out to be an excellent example of the extraordinary properties of RNA molecules to fold into uniquely compact structures. Details of the X-ray structure have greatly stimulated further studies on the folding of the ribozymes into functionally active molecules as well as on the mechanism of RNA catalysis. The ability of the delta ribozymes to carry out general acid-base catalysis by nucleotide side chains has been assumed in two proposed mechanisms of self-cleavage. Recently, considerable progress has been also made in characterizing the catalytic properties of trans-acting ribozyme variants that are potentially attractive tools in the strategy of directed RNA degradation

Altered Expression of Porcine <em>Piwi</em> Genes and piRNA during Development

<div><p>Three <em>Sus scrofa Piwi</em> genes (<em>Piwil1</em>, <em>Piwil2</em> and <em>Piwil4</em>) encoding proteins of 861, 985 and 853 aminoacids, respectively, were cloned and sequenced. Alignment of the Piwi proteins showed the high identity between <em>Sus scrofa</em> and <em>Homo sapiens</em>. Relative transcript abundance of porcine <em>Piwil1</em>, <em>Piwil2</em> and <em>Piwil4</em> genes in testes, ovaries and oocytes derived from sexually immature and mature animals was examined using Real-Time PCR. Expression of the three <em>Piwi</em> mRNAs was proved to be tissue specific and restricted exclusively to the gonads. In testes of adult pigs the highest relative transcript abundance was observed for the <em>Sus scrofa Piwil1</em> gene. On the other hand, in testes of neonatal pigs the <em>Piwil1</em> transcript level was over 2–fold reduced while the level of <em>Piwil2</em> transcript was higher. As regards the expression of the <em>Piwil4</em> transcript, its level was 34-fold elevated in testes of neonatal piglet when compared to adult male. In ovaries of prepubertal and pubertal female pigs transcript abundance of the three <em>Piwi</em> genes was significantly reduced in comparison with testes. However, similarly to testes, in ovaries of neonatal pigs the <em>Piwil</em>2 gene was characterized by the highest relative transcript abundance among the three <em>Piwi</em> genes analysed. In prepubertal and pubertal oocytes <em>Piwil1</em> transcript was the most abundant whereas the expression of <em>Piwil4</em> was undetectable. We also demonstrated that expression of piRNA occurs preferentially in the gonads of adult male and female pigs. Moreover, a piRNA subset isolated from ovaries was 2–3 nucleotides longer than the piRNA from testes.</p> </div

Expression levels of <i>Sus scrofa Piwi</i> family genes in testes of neonatal and adult animals.

<p>Total RNA extracted from the tissues was examined by real-time PCR. Relative transcript abundance of <i>Piwil1</i>, <i>Piwil2</i> and <i>Piwil4</i> genes was normalized to that of <i>β-actin</i> as a reference gene.</p

Tissue specific expression of <i>Piwi</i> genes in testes of adult and neonatal pigs as well as in nine other somatic tissues of neonatal male.

<p>Expression of <i>Piwil1</i>, <i>Piwil2</i> and <i>Piwil4</i> was detected by reverse transcription PCR. Expression of <i>β-actin</i> was used as the inner control. NTC – non template control.</p

Sequence alignment showing the catalytic triad (DDH motif) of mouse Piwil1 and porcine Piwi family proteins.

<p>Catalytic aminoacids are shown in blue.</p