HIV-1 transcript function is controlled in part by twinned transcriptional start site usage, where 5\' capped RNAs beginning with a single guanosine (1G) are preferentially packaged into progeny virions as genomic RNA (gRNA) whereas those beginning with three sequential guanosines (3G) are retained in cells as mRNAs. In 3G transcripts, one of the additional guanosines base pairs with a cytosine located within a conserved 5\' polyA element, resulting in formation of an extended 5\' polyA structure as opposed to the hairpin structure formed in 1G RNAs. To understand how this remodeling influences overall transcript function, we applied in vitro biophysical studies with in-cell genome packaging and competitive translation assays to native and 5\' polyA mutant transcripts generated with promoters that differentially produce 1G or 3G RNAs. We identified mutations that stabilize the 5\' polyA hairpin structure in 3G RNAs, which promote RNA dimerization and Gag binding without sequestering the 5\' cap. None of these 3G transcripts were competitively packaged, confirming that cap exposure is a dominant negative determinant of viral genome packaging. For all RNAs examined, conformations that favored 5\' cap exposure were both poorly packaged and more efficiently translated than those that favored 5\' cap sequestration. We propose that structural plasticity of 5\' polyA and other conserved RNA elements place the 5\' leader on a thermodynamic tipping point for low-energetic (~3 kcal/mol) control of global transcript structure and function.

A novel Apt-LFA has been established for kanamycin based on non-thiolated nucleic acid-modified colloidal gold nanoprobe (AuNPs@polyA-DNA). The improvement in nucleic acid hybridization speed and efficiency was verified by modifying AuNPs with polyA-DNA strands instead of thiolated oligonucleotides (SH-DNA) strands. Moreover, the AuNPs@polyA-DNA was explored to apply in an Apt-LFA. The experimental factors including the concentration of the aptamer, the concentration of SA-DNAT conjugate, the incubation time, and temperature were carefully investigated. In addition, the kanamycin aptamer was modified by extending several bases at its end to modulate the hybridization complementary strand, which was found to significantly improve the performance of Apt-LFA. Under optimal experimental conditions, the Apt-LFA can detect kanamycin in honey with a LOD of 250 ng mL-1 by the naked eyes. A linear range of 50-1250 ng mL-1 was obtained with a LOD of 15 ng mL-1 in honey by a portable reader. The Apt-LFA was successfully applied to the detection of kanamycin in honey with recoveries of 95.1-105.2%.

Plasmodium falciparum is a causative agent of human malaria. Sixty percent of mRNAs from its extremely AT-rich (81%) genome harbor long polyadenosine (polyA) runs within their ORFs, distinguishing the parasite from its hosts and other sequenced organisms. Recent studies indicate polyA runs cause ribosome stalling and frameshifting, triggering mRNA surveillance pathways and attenuating protein synthesis. Here, we show that P. falciparum is an exception to this rule. We demonstrate that both endogenous genes and reporter sequences containing long polyA runs are efficiently and accurately translated in P. falciparum cells. We show that polyA runs do not elicit any response from No Go Decay (NGD) or result in the production of frameshifted proteins. This is in stark contrast to what we observe in human cells or T. thermophila, an organism with similar AT-content. Finally, using stalling reporters we show that Plasmodium cells evolved not to have a fully functional NGD pathway.

The length of untranslated regions at the 3\' end of transcripts (3\'UTRs) is regulated by alternate polyadenylation (APA). 3\'UTRs contain regions that harbor binding motifs for regulatory molecules. However, the mechanisms that coordinate the 3\'UTR length of specific groups of transcripts are not well-understood. We therefore developed a method, CSI-UTR, that models 3\'UTR structure as tandem segments between functional alternative-polyadenylation sites (termed cleavage site intervals-CSIs). This approach facilitated (1) profiling of 3\'UTR isoform expression changes and (2) statistical enrichment of putative regulatory motifs. CSI-UTR analysis is UTR-annotation independent and can interrogate legacy data generated from standard RNA-Seq libraries. CSI-UTR identified a set of CSIs in human and rodent transcriptomes. Analysis of RNA-Seq datasets from neural tissue identified differential expression events within 3\'UTRs not detected by standard gene-based differential expression analyses. Further, in many instances 3\'UTR and CDS from the same gene were regulated differently. This modulation of motifs for RNA-interacting molecules with potential condition-dependent and tissue-specific RNA binding partners near the polyA signal and CSI junction may play a mechanistic role in the specificity of alternative polyadenylation. Source code, CSI BED files and example datasets are available at: https://github.com/UofLBioinformatics/CSI-UTR.

BACKGROUND: Genes encoded in vertebrate mitochondrial DNAs are transcribed as a polycistronic transcript for both strands, which is later processed into individual mRNAs, rRNAs and tRNAs, followed by modifications, such as polyadenylation at the 3\' end of mRNAs. Although mechanisms of the mitochondrial transcription and RNA processing have been extensively studied using some model organisms, structural variability of mitochondrial mRNAs across different groups of vertebrates is poorly understood. We conducted the high-throughput RNA sequencing to identify major polyadenylation sites for mitochondrial mRNAs in the Japanese grass lizard, Takydromus tachydromoides and compared the polyadenylation profiles with those identified similarly for 23 tetrapod species, featuring sauropsid taxa (reptiles and birds).
RESULTS: As compared to the human, a major polyadenylation site for the NADH dehydrogenase subunit 5 mRNA of the grass lizard was located much closer to its stop codon, resulting in considerable truncation of the 3\' untranslated region for the mRNA. Among the other sauropsid taxa, several distinct polyadenylation profiles from the human counterpart were found for different mRNAs. They included various truncations of the 3\' untranslated region for NADH dehydrogenase subunit 5 mRNA in four taxa, bird-specific polyadenylation of the light-strand-transcribed NADH dehydrogenase subunit 6 mRNA, and the combination of the ATP synthase subunit 8/6 mRNA with a neighboring mRNA into a tricistronic mRNA in the side-necked turtle Pelusios castaneus. In the last case of P. castaneus, as well as another example for NADH dehydrogenase subunit 1 mRNAs of some birds, the association between the polyadenylation site change and the gene overlap was highlighted. The variations in the polyadenylation profile were suggested to have arisen repeatedly in diverse sauropsid lineages. Some of them likely occurred in response to gene rearrangements in the mitochondrial DNA but the others not.
CONCLUSIONS: These results demonstrate structural variability of mitochondrial mRNAs in sauropsids. The efficient and comprehensive characterization of the mitochondrial mRNAs will contribute to broaden our understanding of their structural and functional evolution.

This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.Multiple recent publications on RNA sequencing (RNA-seq) have demonstrated the power of next-generation sequencing technologies in whole-transcriptome analysis. Vendor-specific protocols used for RNA library construction often require at least 100 ng total RNA. However, under certain conditions, much less RNA is available for library construction. In these cases, effective transcriptome profiling requires amplification of subnanogram amounts of RNA. Several commercial RNA amplification kits are available for amplification prior to library construction for next-generation sequencing, but these kits have not been comprehensively field evaluated for accuracy and performance of RNA-seq for picogram amounts of RNA. To address this, 4 types of amplification kits were tested with 3 different concentrations, from 5 ng to 50 pg, of a commercially available RNA. Kits were tested at multiple sites to assess reproducibility and ease of use. The human total reference RNA used was spiked with a control pool of RNA molecules in order to further evaluate quantitative recovery of input material. Additional control data sets were generated from libraries constructed following polyA selection or ribosomal depletion using established kits and protocols. cDNA was collected from the different sites, and libraries were synthesized at a single site using established protocols. Sequencing runs were carried out on the Illumina platform. Numerous metrics were compared among the kits and dilutions used. Overall, no single kit appeared to meet all the challenges of small input material. However, it is encouraging that excellent data can be recovered with even the 50 pg input total RNA.

Effect of Zn(2+) ions on the conformation of polyA in cacodilic buffer at pH 7 was investigated by differential UV spectroscopy (DUV) and by thermal denaturation. The shapes of the DUV spectra and melting curves suggest a transition of polyA into a more ordered \"metallized\", possibly double-helical conformation at Zn(2+) concentrations above 3×10(-5) M. A phase diagram of polyA complexes with Zn(2+) was constructed for the temperature range from 20 °C to 95 °C and Zn(2+) concentrations between 10(-5) M and 5×10(-4) M. It was found that the transition of a single strand into the \"metallized\" form is possible only if the length of the disordered single-stranded region becomes larger than a certain critical value, ranging between 98% and 78% as the metal concentration increases from 3×10(-5) to 5×10(-4) M.

Peters plus syndrome is a rare recessive autosomal disorder comprising ocular anterior segment dysgenesis, short stature, hand abnormalities and distinctive facial features. It was related only to mutations in the B3GALTL gene in the 13q12.3 region. In this study, we undertook the first functional analysis of a novel c.597-2 A>G splicing mutation within the B3GALTL gene using an ex-vivo approach. The results showed a complete skipping of exon 8 in the B3GALTL cDNA, which altered the open reading frame of the mutant transcript and generated a PTC within exon 9. This finding potentially elicits the nonsense mRNA to degradation by NMD (nonsense-mediated mRNA decay). The theoretical consequences of splice site mutations, predicted with the bioinformatics tool Human Splice Finder, were investigated and evaluated in relation to ex-vivo results. The findings confirmed the key role played by the B3GALTL gene in typical Peters-plus syndromes and the utility of mRNA analysis to understand the primary impacts of this mutation and the phenotype of the disease.

Analysis of natural host-parasite relationships reveals the evolutionary forces that shape the delicate and unique specificity characteristic of such interactions. The accessory long gland-reservoir complex of the wasp Leptopilina heterotoma (Figitidae) produces venom with virus-like particles. Upon delivery, venom components delay host larval development and completely block host immune responses. The host range of this Drosophila endoparasitoid notably includes the highly-studied model organism, Drosophila melanogaster. Categorization of 827 unigenes, using similarity as an indicator of putative homology, reveals that approximately 25% are novel or classified as hypothetical proteins. Most of the remaining unigenes are related to processes involved in signaling, cell cycle, and cell physiology including detoxification, protein biogenesis, and hormone production. Analysis of L. heterotoma\'s predicted venom gland proteins demonstrates conservation among endo- and ectoparasitoids within the Apocrita (e.g., this wasp and the jewel wasp Nasonia vitripennis) and stinging aculeates (e.g., the honey bee and ants). Enzyme and KEGG pathway profiling predicts that kinases, esterases, and hydrolases may contribute to venom activity in this unique wasp. To our knowledge, this investigation is among the first functional genomic studies for a natural parasitic wasp of Drosophila. Our findings will help explain how L. heterotoma shuts down its hosts\' immunity and shed light on the molecular basis of a natural arms race between these insects.