Originally 2A was characterised in foot-and-mouth disease virus. Site directed mutagenesis identified a C-terminus consensus motif [D(V/I)ExNPGP] and it is proposed that 2A interacts with the exit tunnel of the ribosome in a way that a specific peptide bond is skipped between the last glycine of 2A and the proline of 2B, thus providing a discontinuity in translation, resulting in release of discrete proteins from one single ORF. 2A was also identified in other picornaviruses, positive, single and double-stranded RNA insect viruses and mammalian rotaviruses. A motif present at the C-terminus of the 2A oligopeptide [D(V/I)ExNPGP] is very highly, though not completely conserved . The sequence upstream of this motif shows, however, no apparent conservation between 2As of different viruses. In this study, extensive site-directed mutagenesis were performed on several 2A sequences and a series of ‘hybrid' 2As comprising different consensus motifs juxtaposed with different upstream contexts were created as part of a detailed analysis of the mechanism of 2A-mediated ribosome stalling. The results demonstrated that a minimal region of twenty to twenty-three amino acids interacts with the exit tunnel of the ribosome to bring about a pause in processivity, alter the peptidyl transferase centre geometry and restrict the ribosome A site via two distinctive stalling mechanisms. Other molecular analyses tested here will require further optimisations or alternative methods: a visual method to explore the dynamics of re-initiation of translation from proline codon, purification of the translation-regulating factors and structural resolution of 2A sequences. Previously, cellular 2As were identified in non-LTR retrotransposons of trypanosomes. It is reported here as part of two other cellular organisms Saccoglossus kowalevskii (acorn worm) and Branchiostoma floridae (amphioxus). In the acorn worm, the nucleotides sequences corresponding to 2A motifs were part of the untranslated genome. In amphioxus, three 2A elements were identified in hypothetical proteins, and at the N-terminus of twenty non-LTR retrotransposons.

Indiana University-Purdue University Indianapolis (IUPUI) / Alzheimer disease (AD) results, in part, from the excess accumulation of the amyloid-β peptide (Aβ) as neuritic plaques in the brain. The short Aβ peptide is derived from a large transmembrane precursor protein, APP. Two different proteolytic enzymes, BACE1 and the gamma-secretase complex, are responsible for cleaving Aβ peptide from APP through an intricate processing pathway. Dysregulation of APP and BACE1 levels leading to excess Aβ deposition has been implicated in various forms of AD. Thus, a major goal in this dissertation was to discover novel regulatory pathways that control APP and BACE1 expression as a means to identify novel drug targets central to the Aβ-generating process. MicroRNAs (miRNA) are short, non-coding RNAs that act as post-transcriptional regulators of gene expression through specific interactions with target mRNAs. Global analyses predict that over sixty percent of human transcripts contain evolutionarily conserved miRNA target sites. Therefore, the specific hypothesis tested was that miRNA are relevant regulators of APP and BACE1 expression. In this work, several specific miRNA were identified that regulate APP protein expression (miR-101, miR-153 and miR-346) or BACE1 expression (miR-339-5p). These miRNAs mediated their post-transcriptional effects via interactions with specific target sites in the APP and BACE1 transcripts. Importantly, these miRNA also altered secretion of Aβ peptides in primary human fetal brain cultures. Surprisingly, miR-346 stimulated APP expression via target sites in the APP 5’-UTR. The mechanism of this effect appears to involve other RNA-binding proteins that bind to the APP 5’-UTR. Expression analyses demonstrated that these miRNAs are expressed to varying degrees in the human brain. Notably, miR-101, miR-153 and miR-339-5p are dysregulated in the AD brain at various stages of the disease. The work in this dissertation supports the hypothesis that miRNAs are important regulators of APP and BACE1 expression and are capable of altering Aβ homeostasis. Therefore, these miRNA may possibly serve as novel therapeutic targets for AD.