Andrew D. L. Nelson

Global Analysis of the RNA-Protein Interaction and RNA Secondary Structure Landscapes of the Arabidopsis Nucleus

Posttranscriptional regulation in eukaryotes requires cis- and trans-acting features and factors including RNA secondary structure and RNA-binding proteins (RBPs). However, a comprehensive view of the structural and RBP interaction landscape of nuclear RNAs has yet to be compiled for any organism. Here, we use our ribonuclease-mediated structure and RBP-binding site mapping approaches to globally profile these features in Arabidopsis seedling nuclei in vivo. We reveal anticorrelated patterns of secondary structure and RBP binding throughout nuclear mRNAs that demarcate sites of alternative splicing and polyadenylation. We also uncover a collection of protein-bound sequence motifs, and identify their structural contexts, co-occurrences in transcripts encoding functionally related proteins, and interactions with putative RBPs. Finally, using these motifs, we find that the chloroplast RBP CP29A also interacts with nuclear mRNAs. In total, we provide a simultaneous view of the RNA secondary structure and RBP interaction landscapes in a eukaryotic nucleus.

Dyskerin Is a Component of the Arabidopsis Telomerase RNP Required for Telomere Maintenance

ABSTRACT Dyskerin binds the H/ACA box of human telomerase RNA and is a core telomerase subunit required for RNP biogenesis and enzyme function in vivo. Missense mutations in dyskerin result in dyskeratosis congenita, a complex syndrome characterized by bone marrow failure, telomerase enzyme deficiency, and progressive telomere shortening. Here we demonstrate that dyskerin also contributes to telomere maintenance in Arabidopsis thaliana. We report that both AtNAP57, the Arabidopsis dyskerin homolog, and AtTERT, the telomerase catalytic subunit, accumulate in the plant nucleolus, and AtNAP57 associates with active telomerase RNP particles in an RNA-dependent manner. Furthermore, AtNAP57 interacts in vitro with AtPOT1a, a novel component of Arabidopsis telomerase. Although a null mutation in AtNAP57 is lethal, AtNAP57, like AtTERT, is not haploinsufficient for telomere maintenance in Arabidopsis. However, introduction of an AtNAP57 allele containing a T66A mutation decreased telomerase activity in vitro, disrupted telomere length regulation on individual chromosome ends in vivo, and established a new, shorter telomere length set point. These results imply that T66A NAP57 behaves as a dominant-negative inhibitor of telomerase. We conclude that dyskerin is a conserved component of the telomerase RNP complex in higher eukaryotes that is required for maximal enzyme activity in vivo.

Parameters Affecting Telomere-Mediated Chromosomal Truncation in Arabidopsis

This work describes the development of a robust and quantitative system for elucidating the sequences and trans-acting factors required for de novo telomere formation in Arabidopsis thaliana. We determine that genetic redundancy and the nonhomologous end-joining factors Ku and Lig4 facilitate higher levels of de novo telomere formation. Conversion of a double-strand break into a telomere is a dangerous, potentially lethal event. However, little is known about the mechanism and control of de novo telomere formation (DNTF). DNTF can be instigated by the insertion of a telomere repeat array (TRA) into the host genome, which seeds the formation of a new telomere, resulting in chromosome truncation. Such events are rare and concentrated at chromosome ends. Here, we introduce tetraploid Arabidopsis thaliana as a robust genetic model for DNTF. Transformation of a 2.6-kb TRA into tetraploid plants resulted in a DNTF efficiency of 56%, fivefold higher than in diploid plants and 50-fold higher than in human cells. DNTF events were recovered across the entire genome, indicating that genetic redundancy facilitates recovery of DNTF events. Although TRAs as short as 100 bp seeded new telomeres, these tracts were unstable unless they were extended above a 1-kb size threshold. Unexpectedly, DNTF efficiency increased in plants lacking telomerase, and DNTF rates were lower in plants null for Ku70 or Lig4, components of the nonhomologous end-joining repair pathway. We conclude that multiple competing pathways modulate DNTF, and that tetraploid Arabidopsis will be a powerful model for elucidating the molecular details of these processes.

Protection of Telomeres 1 Is Required for Telomere Integrity in the Moss Physcomitrella patens

In yeast and vertebrates, the essential telomere binding protein POT1 protects chromosome ends, but in Arabidopsis, POT1 proteins have evolved to bind telomerase instead. This study examines the function of POT1 in the moss Physcomitrella patens. The findings show that moss POT1 functions in a manner similar to yeast and vertebrate POT1. Thus, POT1 proteins are evolving very rapidly in plants. In vertebrates, the single-stranded telomeric DNA binding protein Protection of Telomeres 1 (POT1) shields chromosome ends and prevents them from eliciting a DNA damage response. By contrast, Arabidopsis thaliana encodes two divergent full-length POT1 paralogs that do not exhibit telomeric DNA binding in vitro and have evolved to mediate telomerase regulation instead of chromosome end protection. To further investigate the role of POT1 in plants, we established the moss Physcomitrella patens as a new model for telomere biology and a counterpoint to Arabidopsis. The sequence and architecture of the telomere tract is similar in P. patens and Arabidopsis, but P. patens harbors only a single-copy POT1 gene. Unlike At POT1 proteins, Pp POT1 efficiently bound single-stranded telomeric DNA in vitro. Deletion of the P. patens POT1 gene resulted in the rapid onset of severe developmental defects and sterility. Although telomerase activity levels were unperturbed, telomeres were substantially shortened, harbored extended G-overhangs, and engaged in end-to-end fusions. We conclude that the telomere capping function of POT1 is conserved in early diverging land plants but is subsequently lost in Arabidopsis.

ATR cooperates with CTC1 and STN1 to maintain telomeres and genome integrity in Arabidopsis

Telomeres protect chromosome ends from DNA damage. CTC1/STN1/TEN1 (CST), a core telomere-capping complex in plant and vertebrates, suppresses an ATR-dependent DNA damage response in Arabidopsis. Protracted ATR inactivation inhibits telomerase, hastening the onset of telomere dysfunction in CST mutants.

Surprises from the Chromosome Front: Lessons from Arabidopsis on Telomeres and Telomerase

Telomeres serve two vital functions: They act as a buffer against the end-replication problem, and they prevent chromosome ends from being recognized as double-strand DNA (dsDNA) breaks. These functions are orchestrated by the telomerase reverse transcriptase and a variety of telomere protein complexes. Here, we discuss our recent studies with Arabidopsis thaliana that uncovered a new and highly conserved telomere complex called CST (Cdc13/CTC1, STN1, TEN1). Formerly believed to be yeast specific, CST has now been identified as a key component of both plant and vertebrate telomeres, which is essential for genome integrity and stem cell viability. We also describe the unexpected discovery of alternative telomerase ribonucleoprotein complexes in Arabidopsis. Fueled by duplication and diversification of the telomerase RNA subunit and telomerase accessory proteins, these telomerase complexes act in concert to maintain genome stability. In addition to the canonical telomerase enzyme, one of two alternative telomerase ribonucleoprotein (RNP) complexes functions as a novel negative regulator of enzyme activity in response to genotoxic stress. These contributions highlight the immense potential of Arabidopsis in probing the depths of the chromosome end.

Evolution of TERT-interacting lncRNAs: expanding the regulatory landscape of telomerase

Long non-coding RNAs (lncRNAs) evolve rapidly and are functionally diverse. The emergence of new lncRNAs is driven by genome disturbance events, including whole genome duplication, and transposition. One of the few lncRNAs with a conserved role throughout eukaryotes is the telomerase RNA, TER. TER works in concert with the telomerase reverse transcriptase (TERT) to maintain telomeres. Here we discuss recent findings from Arabidopsis thaliana and its relatives illustrating the remarkable evolutionary flexibility within TER and the potential for non-canonical TERT-lncRNA interactions. We highlight the two TERs in A. thaliana. One is a conventional telomerase template. The other lncRNA negatively regulates telomerase activity in response to DNA damage, a function mediated by co-option of a transposable element. In addition, we discuss evidence for multiple independent TER loci throughout the plant family Brassicaceae, and how these loci not only reflect rapid convergent evolution, but also the flexibility of having a lncRNA at the core of telomerase. Lastly, we discuss the propensity for TERT to bind a suite of non-templating lncRNAs, and how such RNAs may facilitate telomerase regulation and off-telomere functions.

A Transposable Element within the Non-canonical Telomerase RNA of Arabidopsis thaliana Modulates Telomerase in Response to DNA Damage

Long noncoding RNAs (lncRNAs) have emerged as critical factors in many biological processes, but little is known about how their regulatory functions evolved. One of the best-studied lncRNAs is TER, the essential RNA template for telomerase reverse transcriptase. We previously showed that Arabidopsis thaliana harbors three TER isoforms: TER1, TER2 and TER2S. TER1 serves as a canonical telomere template, while TER2 is a novel negative regulator of telomerase activity, induced in response to double-strand breaks (DSBs). TER2 contains a 529 nt intervening sequence that is removed along with 36 nt at the RNA 3’ terminus to generate TER2S, an RNA of unknown function. Here we investigate how A. thaliana TER2 acquired its regulatory function. Using data from the 1,001 Arabidopsis genomes project, we report that the intervening sequence within TER2 is derived from a transposable element termed DSB responsive element (DRE). DRE is found in the TER2 loci of most but not all A. thaliana accessions. By analyzing accessions with (TER2) and without DRE (TER2Δ) we demonstrate that this element is responsible for many of the unique properties of TER2, including its enhanced binding to TERT and telomerase inhibitory function. We show that DRE destabilizes TER2, and further that TER2 induction by DNA damage reflects increased RNA stability and not increased transcription. DRE-mediated changes in TER2 stability thus provide a rapid and sensitive switch to fine-tune telomerase enzyme activity. Altogether, our data shows that invasion of the TER2 locus by a small transposon converted this lncRNA into a DNA damage sensor that modulates telomerase enzyme activity in response to genome assault.

Extending the model of Arabidopsis telomere length and composition across Brassicaceae

Telomeres are repetitive TG-rich DNA elements essential for maintaining the stability of genomes and replicative capacity of cells in almost all eukaryotes. Most of what is known about telomeres in plants comes from the angiosperm Arabidopsis thaliana, which has become an important comparative model for telomere biology. Arabidopsis tolerates numerous insults to its genome, many of which are catastrophic or lethal in other eukaryotic systems such as yeast and vertebrates. Despite the importance of Arabidopsis in establishing a model for the structure and regulation of plant telomeres, only a handful of studies have used this information to assay components of telomeres from across land plants, or even among the closest relatives of Arabidopsis in the plant family Brassicaceae. Here, we determined how well Arabidopsis represents Brassicaceae by comparing multiple aspects of telomere biology in species that represent major clades in the family tree. Specifically, we determined the telomeric repeat sequence, measured bulk telomere length, and analyzed variation in telomere length on syntenic chromosome arms. In addition, we used a phylogenetic approach to infer the evolutionary history of putative telomere-binding proteins, CTC1, STN1, TEN1 (CST), telomere repeat-binding factor like (TRFL), and single Myb histone (SMH). Our analyses revealed conservation of the telomeric DNA repeat sequence, but considerable variation in telomere length among the sampled species, even in comparisons of syntenic chromosome arms. We also found that the single-stranded and double-stranded telomeric DNA-binding complexes CST and TRFL, respectively, differ in their pattern of gene duplication and loss. The TRFL and SMH gene families have undergone numerous duplication events, and these duplicate copies are often retained in the genome. In contrast, CST components occur as single-copy genes in all sampled genomes, even in species that experienced recent whole genome duplication events. Taken together, our results place the Arabidopsis model in the context of other species in Brassicaceae, making the family the best characterized plant group in regard to telomere architecture.

A genomic analysis of factors driving lincRNA diversification: lessons from plants

Transcriptomic analyses from across eukaryotes indicate that most of the genome is transcribed at some point in the developmental trajectory of an organism. One class of these transcripts is termed long intergenic noncoding RNAs (lincRNAs). Recently, attention has focused on understanding the evolutionary dynamics of lincRNAs, particularly their conservation within genomes. Here, we take a comparative genomic and phylogenetic approach to uncover factors influencing lincRNA emergence and persistence in the plant family Brassicaceae, to which Arabidopsis thaliana belongs. We searched 10 genomes across the family for evidence of > 5000 lincRNA loci from A. thaliana. From loci conserved in the genomes of multiple species, we built alignments and inferred phylogeny. We then used gene tree/species tree reconciliation to examine the duplication history and timing of emergence of these loci. Emergence of lincRNA loci appears to be linked to local duplication events, but, surprisingly, not whole genome duplication events (WGD), or transposable elements. Interestingly, WGD events are associated with the loss of loci for species having undergone relatively recent polyploidy. Lastly, we identify 1180 loci of the 6480 previously annotated A. thaliana lincRNAs (18%) with elevated levels of conservation. These conserved lincRNAs show higher expression, and are enriched for stress-responsiveness and cis-regulatory motifs known as conserved noncoding sequences (CNSs). These data highlight potential functional pathways and suggest that CNSs may regulate neighboring genes at both the genomic and transcriptomic level. In sum, we provide insight into processes that may influence lincRNA diversification by providing an evolutionary context for previously annotated lincRNAs.