No abstract

We have investigated the secondary structure of peach latent mosaic viroid (PLMVd) in solution, and we present here the first description of the structure of a branched viroid in solution. Different PLMVd transcripts of plus polarity were produced by using the circularly permuted RNA method and the exploitation of RNA internal secondary structure to position the 5 and 3 termini and studied by nuclease mapping and binding shift assays using DNA and RNA oligonucleotides. We show that PLMVd folds into a complex, branched secondary structure. In general, this structure is similar to that reported previously, which was based on sequence comparison and computer modelling. The structural microheterogeneity is apparently limited to only some small domains. More importantly, this structure includes a novel pseudoknot that is conserved in all PLMVd isolates and seems to allow folding into a very compact form. This pseudoknot is also found in chrysanthemum chlorotic mottle viroid, suggesting that it is a unique feature of the viroid members of the PLMVd subgroup.Viroids are small (ϳ300 nucleotides), single-stranded, circular RNAs that infect higher plants, causing significant losses in the agricultural industry (see references 7 and 13 for reviews). Viroids have been classified in two groups (groups A and B) based primarily on whether or not they possess five typical structural domains found in the group B viroids (7). Further division among the group B members depends on the sequence and length of the conserved central region. Viroids that do not possess any kind of sequence or structural similarity with the group B viroids have been classified as belonging to group A. The viroids from this group possess self-cleaving hammerhead motifs that are crucial for their replication via a rolling circle mechanism.The group A viroids include the avocado sunblotch viroid (ASBVd), the peach latent mosaic viroid (PLMVd), and the chrysanthemum chlorotic mottle viroid (CChMVd) (10). Both PLMVd and CChMVd have been proposed to adopt branched secondary structures (Fig. 1A) instead of the rod-like ones proposed for most viroids, including ASBVd (10). The unusual conformations of PLMVd and CChMVd are supported by their insolubility in 2 M lithium chloride, whereas ASBVd and a number of non-self-cleaving viroids (i.e., the group B viroids) are soluble in this high salt solution (10). In general, secondary structures of viroids are predicted using computer software and are useful for the formulation of hypotheses on the structure-function relationships of these RNA molecules (4). Characterization of biological structures in vitro as well as in vivo is obviously more accurate for elucidating the structure-function relationship. The only secondary structure of a viroid that has been extensively studied in solution is that of the potato spindle tuber viroid (8). This group B species, which was shown to adopt a rod-like shape in solution, is responsible for most of our knowledge of the biology of viroids.In order to determine the secondary struct...

HREM improves our understanding and allows precise characterization of esophageal dysmotility in patients who have undergone EA repair.

HNF4α is a nuclear receptor produced as 12 isoforms from two promoters by alternative splicing. To characterize the transcriptional capacities of all 12 HNF4α isoforms, stable lines expressing each isoform were generated. The entire transcriptome associated with each isoform was analyzed as well as their respective interacting proteome. Major differences were noted in the transcriptional function of these isoforms. The α1 and α2 isoforms were the strongest regulators of gene expression whereas the α3 isoform exhibited significantly reduced activity. The α4, α5, and α6 isoforms, which use an alternative first exon, were characterized for the first time, and showed a greatly reduced transcriptional potential with an inability to recognize the consensus response element of HNF4α. Several transcription factors and coregulators were identified as potential specific partners for certain HNF4α isoforms. An analysis integrating the vast amount of omics data enabled the identification of transcriptional regulatory mechanisms specific to certain HNF4α isoforms, hence demonstrating the importance of considering all isoforms given their seemingly diverse functions.

Pseudogenes are mutated copies of protein-coding genes that cannot be translated into proteins, but a small subset of pseudogenes has been detected at the protein level. Although ubiquitin pseudogenes represent one of the most abundant pseudogene families in many organisms, little is known about their expression and signaling potential. By re-analyzing public RNA-sequencing and proteomics datasets, we here provide evidence for the expression of several ubiquitin pseudogenes including UBB pseudogene 4 (UBBP4), which encodes Ub KEKS (Q2K, K33E, Q49K, N60S). The functional consequences of Ub KEKS conjugation appear to differ from canonical ubiquitylation. Quantitative proteomics shows that Ub KEKS modifies specific proteins including lamins. Knockout of UBBP4 results in slower cell division, and accumulation of lamin A within the nucleolus. Our work suggests that a subset of proteins reported as ubiquitin targets may instead be modified by ubiquitin variants that are the products of wrongly annotated pseudogenes and induce different functional effects.