The Long-read RNA-Seq Genome Annotation Assessment Project Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. Using different protocols and sequencing platforms, the consortium generated over 427 million long-read sequences from complementary DNA and direct RNA datasets, encompassing human, mouse and manatee species. Developers utilized these data to address challenges in transcript isoform detection, quantification and de novo transcript detection. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. Incorporating additional orthogonal data and replicate samples is advised when aiming to detect rare and novel transcripts or using reference-free approaches. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.

Citrus greening or huanglongbing (HLB) is a devastating disease of citrus. HLB is associated with the phloem-limited fastidious prokaryotic α-proteobacterium ‘Candidatus Liberibacter spp.’ In this report, we used sweet orange (Citrus sinensis) leaf tissue infected with ‘Ca. Liberibacter asiaticus’ and compared this with healthy controls. Investigation of the host response was examined with citrus microarray hybridization based on 33,879 expressed sequence tag sequences from several citrus species and hybrids. The microarray analysis indicated that HLB infection significantly affected expression of 624 genes whose encoded proteins were categorized according to function. The categories included genes associated with sugar metabolism, plant defense, phytohormone, and cell wall metabolism, as well as 14 other gene categories. The anatomical analyses indicated that HLB bacterium infection caused phloem disruption, sucrose accumulation, and plugged sieve pores. The up-regulation of three key starch biosynthetic genes including ADP-glucose pyrophosphorylase, starch synthase, granule-bound starch synthase and starch debranching enzyme likely contributed to accumulation of starch in HLB-affected leaves. The HLB-associated phloem blockage resulted from the plugged sieve pores rather than the HLB bacterial aggregates since ‘Ca. Liberibacter asiaticus’ does not form aggregate in citrus. The up-regulation of pp2 gene is related to callose deposition to plug the sieve pores in HLB-affected plants.

Adiponutrin and a related protein, adipocyte triglyceride lipase (ATGL; also known as Desnutrin), were recently described as adipocyte-specific proteins with lipid hydrolase activity. Using bioinformatics, we identified three additional Adiponutrin family members (GS2, GS2-Like, and PNPLA1). Here, we report on the expression, regulation, and activity of GS2 and GS2-Like compared with Adiponutrin and Desnutrin/ATGL. GS2-Like is expressed and regulated in a manner similar to Adiponutrin; however, the absolute levels of mRNA are significantly lower than those of Adiponutrin or Desnutrin/ATGL. GS2 transcripts were identified only in humans and are highly expressed in adipose as well as other tissues. All four proteins show lipase activity in vitro, which is dependent on the presence of the active site serine for Adiponutrin, Desnutrin/ATGL, and GS2. Overexpression of Desnutrin/ATGL, GS2, and GS2-Like, but not Adiponutrin, decreases intracellular triglyceride levels. This is consistent with a function for Desnutrin/ATGL, GS2, and GS2-Like in lipolysis, but not for Adiponutrin. Consistent with previously reported data, Desnutrin/ATGL is upregulated by fasting in adipose tissue, whereas Adiponutrin is downregulated. Additionally, Adiponutrin and GS2-Like, but not Desnutrin/ATGL, are strongly induced in the liver of ob/ob mice.

Paxillin is a 68-kDa focal adhesion protein that is phosphorylated on tyrosine residues in fibroblasts in response to transformation by v-src, treatment with platelet-derived growth factor, or cross-linking of integrins. Paxillin has been shown to have binding sites for the SH3 domain of Src and the SH2 domain of Crk in vitro and to coprecipitate with two other focal adhesion proteins, vinculin and focal adhesion kinase (p125^fak). After preliminary studies showed that paxillin was a substrate for the hematopoietic oncogene p210^BCR/ABL, we investigated the role of this protein in hematopoietic cell transformation and signal transduction. A full-length cDNA encoding human paxillin was cloned, revealing multiple protein domains, including four tandem LIM domains, a proline-rich domain containing a consensus SH3 binding site, and three potential Crk-SH2 binding sites. The paxillin gene was localized to chromosome 12q24 by fluorescence in situ hybridization analysis. A chicken paxillin cDNA was also cloned and is predicted to encode a protein approximately 90% identical to human paxil-lin. Paxillin coprecipitated with p210^BCR/ABL and mul-tiple other cellular proteins in myeloid cell lines, suggesting the formation of multimeric complexes. In normal hematopoietic cells and myeloid cell lines, tyrosine phosphorylation of paxillin and coprecipitation with other cellular proteins was rapidly and transiently induced by interleukin-3 and several other hematopoietic growth factors. The predicted structure of paxillin implicates this molecule in protein-protein interactions involved in signal transduction from growth factor receptors and the BCR/ABL oncogene fusion protein to the cytoskeleton.

Acyl-CoA:glycerol-3-phosphate acyltransferase (GPAT) catalyzes the first step during de novo synthesis of triacylglycerol. It has been well recognized that mammals possess multiple enzymatically distinct proteins with GPAT activity. Although the mitochondrial-associated GPAT has been cloned and extensively characterized, the molecular identity of the endoplasmic reticulum (ER)-associated GPAT, which accounts for the majority of total GPAT activity in most tissues, has remained elusive. Here we report the identification of genes encoding human and mouse ER-associated GPAT (termed GPAT3). GPAT3 is a member of the acyltransferase family predominantly expressed in tissues characterized by active lipid metabolism, such as adipose tissue, small intestine, kidney, and heart. Ectopic expression of GPAT3 leads to a significant increase in N -ethylmaleimide-sensitive GPAT activity, whereas acyltransferase activity toward a variety of other lysophospholipids, as well as neutral lipid substrates, is not altered. Overexpression of GPAT3 in mammalian cells results in increased triacylglycerol, but not phospholipid, formation. GPAT3 is localized to the ER when overexpressed in COS-7 cells. GPAT3 mRNA is dramatically up-regulated during adipocyte differentiation, is reciprocally regulated in adipose tissue and liver of ob/ob mice, and is up-regulated in mice treated with a peroxisome proliferator-activated receptor γ (PPARγ) agonist. A substantial loss of GPAT activity in 3T3-L1 adipocytes was achieved by reducing GPAT3 mRNA levels through GPAT3-specific siRNA knockdown. These findings identify GPAT3 as a previously uncharacterized triacylglycerol biosynthetic enzyme. Similar to other lipogenic enzymes, GPAT3 may be useful as a target for the treatment of obesity.