TDP-43 is a predominantly nuclear RNA-binding protein that forms inclusion bodies in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The mRNA targets of TDP-43 in the human brain and its role in RNA processing are largely unknown. Using individual-nucleotide resolution UV-crosslinking and immunoprecipitation (iCLIP), we demonstrated that TDP-43 preferentially binds long clusters of UG-rich sequences in vivo. Analysis of TDP-43 RNA binding in FTLD-TDP brains revealed the greatest increases in binding to MALAT1 and NEAT1 non-coding RNAs. We also showed that TDP-43 binding on pre-mRNAs influences alternative splicing in a similar position-dependent manner to Nova proteins. In addition, we identified unusually long clusters of TDP-43 binding at deep intronic positions downstream of silenced exons. A significant proportion of alternative mRNA isoforms regulated by TDP-43 encode proteins that regulate neuronal development or are implicated in neurological diseases, highlighting the importance of TDP-43 for splicing regulation in the brain.

Snake venoms are rich sources of serine proteinase inhibitors that are members of the Kunitz/BPTI (bovine pancreatic trypsin inhibitor) family. However, only a few of their gene sequences have been determined from snakes. We therefore cloned the cDNAs for the trypsin and chymotrypsin inhibitors from a Vipera ammodytes venom gland cDNA library. Phylogenetic analysis of these and other snake Kunitz/BPTI homologs shows the presence of three clusters, where sequences cluster by functional role. Analysis of the nucleotide sequences from the snake Kunitz/BPTI family shows that positive Darwinian selection was operating on the highly conserved BPTI fold, indicating that this family evolved by gene duplication and rapid diversi¢cation.

This study examined the evolutionary dynamics of Bov-B LINEs in vertebrates and the evolution of the RTE clade of non-LTR retrotransposons. The first full-length reptilian Bov-B LINE element is described; it is 3.2 kb in length, with a structural organization typical of the RTE clade of non-LTR retrotransposons. The long-term evolution of Bov-B LINEs was studied in 10 species of Squamata by analysis of a PCR-amplified 1.8-kb fragment encoding part of apurinic/apyrimidinic endonuclease, the intervening domain, and the palm/fingers subdomain of reverse transcriptase. A very high level of conservation in Squamata Bov-B long interspersed nuclear elements has been found, reaching 86% identity in the nearly 600 amino acids of ORF2. The same level of conservation exists between the ancestral snake lineage and Ruminantia. Such a high level is exceptional when compared with the level of conservation observed in nuclear and mitochondrial proteins and in other transposable elements. The RTE clade has been found to be much more widely distributed than previously thought, and novel representatives have been discovered in plants, brown algae, annelids, crustaceans, mollusks, echinoderms, and teleost fishes. Evolutionary relationships in the RTE clade were deduced at the amino acid level from three separate regions of ORF2. By using different independent methods, including the divergence-versus-age analysis, several examples of horizontal transfer in the RTE clade were recognized, with important implications for the existence of HT in non-LTR retrotransposons.

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