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%.

The aptamer-based lateral flow assay strips (Apt-LFAs) have shown promising application prospects in the detection of small molecules. The general principle of Apt-LFAs used for the detection of small molecules is based on the target-induced dissociation (TID) competitive binding among the aptamer, target and gold nanoparticle (AuNP)-complementary DNA (cDNA) nanoprobes. One of the most important components in this device is AuNP-cDNA nanoprobe, which has strong effect on the sensitivity and specificity of Apt-LFAs. In this report, we designed an AuNPs@polyA-cDNA nanoprobe, which consists of a poly adenine (polyA) anchor blocker, a partial complementary DNA fragment to aptamer strand (cDNAa) and complementary DNA fragment to control DNA strand (cDNAc), for rapid detection of acetamiprid. cDNAa of AuNPs@polyA-cDNA nanoprobe was carefully investigated in terms of the hybridization site and length with the aptamer. A specific cDNAa sequence containing key binding bases of acetamiprid aptamer was obtained and verified by molecular docking analysis. After systematic optimization, the Apt-LFA was able to detect a minimum concentration of 0.33 ng mL-1 acetamiprid. The Apt-LFA was successfully applied to detect spiked acetamiprid in tomato and rape samples with the recoveries ranged from 94 to 106%. Based on the strong versatility and verified molecular interaction mechanism, the design strategy could be extended to develop various Apt-LFAs for other small molecules.

Herein, we report rapid electrochemical detection of miRNA let-7a based on a DNA probe consisting of a polyA and Fc-co-labeled harpin structure (the polyA-H probe). The polyA-H probe could be facilely immobilized on Au surfaces through the interactions between polyA and Au, followed by its pre-hybridization with a single strand (S1). The probe\'s surface density could be optimized for minimizing steric hindrance via changing the polyA block length. The target let-7a could be rapidly amplified via loop-mediated isothermal amplification (LAMP) with four simplified primers, followed by inducing the formation of dimeric i-motif (DIM) structure via H+-induced rapid folding of two C-rich sequences of motif strand 1 and strand 2. It was found that, after introducing the as-formed DIM to hybridize the S1, the immobilized polyA20-H probe could rapidly revert to its hairpin structure, sending out a turn-on electrochemical signal of the Fc. The total time for detecting the let-7a was around 80 min, obviously less than that of most of electrochemical DNA sensors reported previously. The biosensor showed a linear relationship of the current response to the let-7a in the range of 10 fM to 50 nM with a limit of detection (LOD) of 5.1 fM. Our biosensors were further tested using human serum spiked with the let-7a and the extracts of the breast adenocarcinoma cells spiked with and without the let-7a, respectively. Satisfied results were obtained. This study shows a potential promising future of development of electrochemical biosensors for rapid detection of miRNAs in the application of clinical practice.

mRNA binding proteins regulate gene expression by controlling the processing, localization, decay, and translation of messenger RNAs (mRNAs). To fully understand these mechanisms of posttranscriptional gene regulation, it is necessary to identify the complete set of mRNA binding proteins. In recent years, several assays have been developed to accomplish this goal in a wide variety of organisms. This work describes a method for the systematic identification of mRNA binding proteins in Saccharomyces cerevisiae. This method applies in vivo UV cross-linking, affinity pull-down of polyA(+) mRNAs, and analysis by mass spectrometry to identify proteins that directly bind to mRNAs.

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.

The maturation, localization, stability, and translation of messenger RNAs (mRNAs) are regulated by a wide variety of mRNA-binding proteins. Identification of the complete set of mRNA-binding proteins is a key step in understanding the regulation of gene expression. Herein, we describe a method for identifying yeast mRNA-binding proteins in a systematic manner using UV crosslinking, purification of polyA(+) mRNAs under denaturing conditions, and mass spectrometry to identify covalently bound proteins.

In this work, we investigate the compaction activity of a sequential alpha,epsilon-peptide composed of l-lysines towards two RNA targets, in view of its possible pharmaceutical application in RNA-targeting and RNA delivery. The basic oligolysine, object of the present study, proved not only to be efficient in compacting the single-stranded polyA RNA, but also to strongly interact with the polyA·polyU complex, as evidenced by CD-binding and UV-melting experiments. In particular, the marked differences in the CD spectra of the RNA targets upon addition of the peptide, as well as the different UV melting behaviour for the polyA·polyU complex in the presence and absence of the peptide, sustain the hypothesis of a strong RNA compaction capacity of the alpha,epsilon-oligolysine. Finally, by using HPLC analysis, we found a good resistance of the peptide against the lytic action of human serum, an important requirement in view of in vitro/in vivo biological assays.