Identifying Long Noncoding RNAs and Examining Their Functional Roles in Brassicaceae

Tremendous progress has been made over the last century characterizing the structure, function, and evolutionary dynamics of protein-coding genes. Over the last three decades, biologists have sequenced the genomes of a variety of organisms, and it quickly became apparent that a relatively small portion of most genomes encode protein-coding genes. We now know that the non-protein-coding space contains repetitive sequences along with non-coding RNAs. Many of these non-coding RNAs encode housekeeping transcripts such as: ribosomal RNAs, transfer RNAs, small nucleolar RNAs, and small nuclear RNAs. However, the remaining transcripts include regulatory RNAs that have become the focus of intense investigation over the past two decades. These include the microRNAs and the small interfering RNAs which both act to knock down gene expression. More recently, a class of transcripts called long non-coding RNAs (lncRNAs) have been interrogated. One well described, functional lncRNA is the telomerase RNA which is necessary for genome stability via the maintenance of chromosome ends following DNA replication (Dew-Budd et al. 2020). While lncRNAs have been known for more than two decades, their functions have remained enigmatic; there are few functionally characterized lncRNAs. However, lncRNAs that have been characterized, especially in mammalian systems, appear to fine tune a plethora of developmental and regulatory processes, along with responses to environmental cues. LncRNAs are frequently expressed at low levels and in specific tissues; they are often poorly conserved among closely related species (Palos et al. 2021). As high-throughput RNA-sequencing becomes more widely available and used, researchers have employed the resulting data to identify lncRNAs en masse. For mammals, a variety of high-quality resources have been used to generate informative annotations to allow for improved hypothesis generation. These resources include GENCODE (Frankish et al. 2021), coupled with lncRNA identification efforts by Cabili et al. (2011) and Guttman et al. (2009). However, while identification and annotation of lncRNAs have been performed in a variety of plant species, they are generally focused on specific tissues or stresses with non-uniform methods for identifying lncRNAs. This inconsistency likely contributes to the paucity of functionally characterized lncRNAs in plants. In this dissertation, I present three chapters in which I used comparative genomics and transcriptomics to increase our understanding of where lncRNAs are located inside cells, what they look like, what they are doing, and how they are conserved. First, I perform a comprehensive annotation effort across four Brassicaceae species using publicly available RNA-seq data to identify lncRNAs, study their expression patterns, epigenetic characteristics, and evolutionary relationships. This work identified thousands of candidate lncRNAs for follow up study and provided a suite of informative characteristics for many. Next, I studied how prolonged salt stress affects lncRNA expression and accumulation in purified nuclei as well as whole cells in three Brassicaceae. Finally, I performed an evolutionary analysis on a previously characterized lncRNA, HIDDEN TREASURE 1, showing that it has undergone extensive duplication and loss across flowering plants. In sum, my work contributes to the growing field of plant lncRNA biology and provides substantive resources for future lncRNA research