SINE Mobilization Analysis to Identify Structural Characteristic That Assists With Cytoplasmic Mobiliziation To the Ribosome

Short Interspersed Nuclear Elements (SINEs) are non-autonomous mobile elements present in eukaryotic genomes. SINEs are mobilized in the form of a transcribed RNA intermediate in a `copy and paste’ process referred to as retrotransposition, by making use of the enzymes encoded by an autonomous element pair, or a Long Interspersed Nuclear Element (LINE). In turn, the active LINE is able to mobilize its own mRNA (termed retrotransposition in cis) or a SINE RNA (in trans). SINE insertions have been linked to not just genetic diversity but also naturally and artificially selected phenotypes in humans and non-human mammals. Within these host genomes, SINEs differ in their activities and evolutionary histories. Unlike a LINE, which contains an RNA II polymerase promoter, a SINE is known for its internal polymerase III promoter which has contributed to its structure and an advantage in utilizing the mobile proteins from LINE, for example in the human Alu by attaching onto the ribosome via conserved structure involving signal recognition particles (SRP). The active and most predominant SINE in human genomes, Alu, is derived from 7SL RNA; the active SINE in canine genomes, SINE_Cf is derived from a tRNALys, and active SINEs in murine genomes, B1 and B2, are derived from either 7SL and tRNA, respectively, each of the above in an evolutionary independent manner. Members of the currently active Alu/LINE pair, of the specific AluY (i.e., for `young’) and L1Hs (`L1’ for `active’ and “Hs’ for `human specific’) have been very well studied and their mobilization detailed in an elegant model. Alu RNA adopts the conserved structure and cellular binding partners as it’s ancestral 7SL RNA molecule, SRP9/14, which directs the Alu intermediate to the ribosome. In the currently understood Alu/L1Hs model, while docked on the ribosome, the Alu RNA intermediate `piggybacks’ the translated L1Hs ORF2p at the ribosome, which functions to transport the intermediate to the nucleus and reverse transcribe it into a new locus as double-stranded DNA. This process is referred to as retrotransposition. Alu retrotransposition has been shown in a cellular ex vivo assay using the SINE element marked with a reporter gene, neoR, in which mobilization is assessed by the relative presence of neoR cellular foci present following L1Hs-driven retrotransposition in trans. We hypothesize by exploring relative activities of non-Alu SINE elements, we will be able to identify structural characteristics and potential sites of host interaction involved in mobilization and ribosome association. Using the described ex vivo assay, we have cloned tRNA SINE elements and preliminarily assessed their mobilization. This work should contribute to our understanding of the evolution and patterns of ribosomal access of non-7SL SINEs in mammalian genomes.