Biology subjects

Reddy, A.

Publications and source records attributed to Reddy, A..

2 recordsLinked to original sources

bioRxiv2017

Development of an in vitro pre-mRNA splicing assay using plant nuclear extract

BackgroundPre-mRNA splicing is an essential post-transcriptional process in all eukaryotes. In vitro splicing systems using nuclear or cytoplasmic extracts from mammalian cells, yeast, and Drosophila have provided a wealth of mechanistic insights into assembly and composition of the spliceosome, splicing regulatory proteins and mechanisms of pre-mRNA splicing in non-plant systems. The lack of an in vitro splicing system prepared from plant cells has been a major limitation in splicing research in plants.\n\nResultsHere we report an in vitro splicing assay system using plant nuclear extract. Several lines of evidence indicate that nuclear extract (NE) derived from Arabidopsis seedlings can convert pre-mRNA substrate (LHCB3) into a spliced product. These include: i) generation of an RNA product that corresponds to the size of expected mRNA, ii) a junction-mapping assay using S1 nuclease revealed that the two exons are spliced together, iii) the reaction conditions are similar to those found with non-plant extracts and iv) finally mutations in conserved donor and acceptor sites abolished the production of the spliced product.\n\nConclusionsThis first report on the plant in vitro splicing assay opens new avenues to investigate plant spliceosome assembly and composition, and splicing regulatory mechanisms specific to plants.

plant biology↗

bioRxiv2017

Id proteins suppress E2A-driven innate-like T cell development prior to TCR selection

Id proteins have been shown to promote the differentiation of conventional {beta} and {gamma}{delta}T cells, and to suppress the expansion of invariant Natural Killer T (iNKT) cells and innate-like {gamma}{delta}NKT within their respective cell lineages. However, it remains to be determined whether Id proteins regulate lineage specification in developing T cells that give rise to these distinct cell fates. Here we report that in the absence of Id2 and Id3 proteins, E2A prematurely activates genes critical for the iNKT cell lineage prior to TCR expression. Enhanced iNKT development in Id3-deficient mice lacking {gamma}{delta} NKT cells suggests that Id3 regulates the lineage competition between these populations. RNA-Seq analysis establishes E2A as the transcriptional regulator of both iNKT and {gamma}{delta}NKT development. In the absence of pre-TCR signaling, Id2/Id3 deletion gives rise to a large population of iNKT cells and a unique innate-like DP population, despite the block in conventional {beta} T cell development. The transcriptional profile of these unique DP cells reflects enrichment of innate-like signature genes, including PLZF (Zbtb16) and Granzyme A (Gzma). Results from these genetic models and genome-wide analyses suggest that Id proteins suppress E2A-driven innate-like T cell programs prior to TCR selection to enforce predominance of conventional T cells.

immunology↗