Sense Strand Research Articles

Controlling Gene-Silencing with Azobenzene-Containing siRNAs (siRNAzos).

This article contains the detailed biophysical characterization, biological testing, and photo-switching protocols of azobenzene containing siRNAs (siRNAzos), which have photoswitchable properties that can be controlled with light. First, the siRNAzos are characterized by annealing the sense and anti-sense strands together and then measuring the circular dichroism (CD) profile, and the melting temperatures (Tm ) of the duplexes. Second, the biological testing of the siRNAzos in cell culture is done to determine their gene silencing efficacy. Finally, their gene-silencing activities are measured after exposure to ultraviolet (UV) light in order to inactivate the siRNAzo, and then broadband visible light, which re-activates the siRNAzo. This inactivation/reactivation protocol can be done in real time, and is reversible and robust and can be performed multiple times on the same sample if desired. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Bio-physical characterization of siRNAzo duplexes Basic Protocol 2: Evaluation of azobenzene gene-silencing using Firefly Luciferase Basic Protocol 3: Evaluation of azobenzene gene-silencing using reverse transcriptase-polymerase chain reaction (RT-PCR).

Grass carp reovirus encoding circular RNAs with antiviral activity

Circular RNAs (circRNAs) are novel, single-stranded, non-coding RNAs that play important roles in many biological processes. CircRNAs have not only been identified in eukaryotes, they have also been identified in some DNA viruses. Grass carp reovirus (GCRV) is a dsRNA virus; the genome of this virus contains 11 segmented dsRNAs. Infection of grass carp with GCRV leads to haemorrhagic disease, one of the most serious diseases in the grass carp aquaculture industry. To investigate the expression of circRNAs in this dsRNA virus, and their potential role in viral infection, we performed transcriptome analysis of GCRV-infected Ctenopharyngodon idelluskidney (CIK) cells. In total, 32 GCRV-derived circRNAs (vcircRNAs) were identified using a back-splicing strategy. Thirty-one vcircRNAs were derived from antisense strands of dsRNA segments; only one vcircRNA was derived from the sense strand. Our analysis predicted that some vcircRNAs, with the same 5′ terminal and variable 3′ terminal regions, were generated via alternative splicing events. The expression levels of vcircRNAs increased in CIK cells following GCRV infection. Competing endogenous RNA analysis further showed that the effects of vcircRNAs on host or viral genes may act via miRNAs. Furthermore, some vcircRNAs, that were transfected into cells via in vitro syntheses, caused a significant increase in cell apoptosis when compared with linear sequences of vcircRNAs or circRNA-GFP (as controls). Finally, we found that the expression levels of GCRV genes in CIK cells were significantly suppressed following the transfection of vcircRNAs. Collectively, our findings indicate that vcircRNAs are a novel form of viral transcript that are expressed in virus-infected cells. Our data also indicate that vcircRNAs may sequester the functions of host miRNAs or viral miRNAs in order to regulate genes in the host or virus to inhibit viral infection.

Characterization of a Strain of Malva Vein Clearing Virus in Alcea rosea via Deep Sequencing.

Malva vein clearing virus (MVCV) is a member of the Potyvirus species, and has a negative impact on the aesthetic development of Alcea rosea. It was first reported in Germany in 1957, but its complete genome sequence data are still scarce. In the present work, A. rosea leaves with vein-clearing and mosaic symptoms were sampled and analyzed with small RNA deep sequencing. By denovo assembly the raw sequences of virus-derived small interfering RNAs (vsiRs) and whole genome amplification of malva vein cleaning virus SX strain (MVCV-SX) by specific primers targeting identified contig gaps, the full-length genome sequences (9,645 nucleotides) of MVCV-SX were characterized, constituting of an open reading frame that is long enough to encode 3,096 amino acids. Phylogenetic analysis showed that MVCV-SX was clustered with euphorbia ringspot virus and yam mosaic virus. Further analyses of the vsiR profiles revealed that the most abundant MVCV-vsiRs were between 21 and 22 nucleotides in length and a strong bias was found for “A” and “U” at the 5′-terminal residue. The results of polarity assessment indicated that the amount of sense strand was almost equal to that of the antisense strand in MVCV-vsiRs, and the main hot-spot region in MVCV-SX genome was found at cylindrical inclusion. In conclusion, our findings could provide new insights into the RNA silencing-mediated host defence mechanism in A. rosea infected with MVCV-SX, and offer a basis for the prevention and treatment of this virus disease.

Synthesis, chirality-dependent conformational and biological properties of siRNAs containing 5'-(R)- and 5'-(S)-C-methyl-guanosine.

Various chemical modifications have been identified that enhance potency of small interfering RNAs (siRNAs) and that reduce off-target effects, immune stimulation, and toxicities of metabolites of these therapeutic agents. We previously described 5′-C-methyl pyrimidine nucleotides also modified at the 2′ position of the sugar. Here, we describe the synthesis of 2′-position unmodified 5′-(R)- and 5′-(S)-C-methyl guanosine and evaluation of these nucleotides in the context of siRNA. The (R) isomer provided protection from 5′ exonuclease and the (S) isomer provided protection from 3′ exonuclease in the context of a terminally modified oligonucleotide. siRNA potency was maintained when these modifications were incorporated at the tested positions of sense and antisense strands. Moreover, the corresponding 5′ triphosphates were not substrates for mitochondrial DNA polymerase. Models generated based on crystal structures of 5′ and 3′ exonuclease oligonucleotide complexes with 5′-(R)- and 5′-(S)-C-methyl substituents attached to the 5′- and 3′-terminal nucleotides, respectively, provided insight into the origins of the observed protections. Structural properties of 5′-(R)-C-methyl guanosine incorporated into an RNA octamer were analysed by X-ray crystallography, and the structure explains the loss in duplex thermal stability for the (R) isomer compared with the (S) isomer. Finally, the effect of 5′-C-methylation on endoribonuclease activity has been explained.

Highly homologous mouse Cyp2a4 and Cyp2a5 genes are differentially expressed in the liver and both express long non-coding antisense RNAs.

The mammalian Cytochrome P450 (Cyp) gene superfamily encodes enzymes involved in numerous metabolic pathways and are frequently expressed in the liver. Despite the remarkably high sequence similarity of Cyp2a4 and Cyp2a5 genes and their surrounding genomic regions, they exhibit differences in expression in the adult mouse liver. For example, Cyp2a4 is highly female-biased whereas Cyp2a5 is only moderately female-biased and Cyp2a4, but not Cyp2a5, is activated in liver cancer. We hypothesized that the limited sequence differences may help us identify the basis for this differential expression. An antisense expressed sequence tag had been uniquely annotated to the Cyp2a4 gene which led us to investigate this transcript as a possible regulator of this gene. We characterized the full-length antisense transcript and also discovered a similar transcript in the Cyp2a5 gene. These transcripts are nuclear long noncoding RNAs that are expressed similarly to their sense mRNA counterparts. This includes the sex-biased and liver tumor differences seen between the Cyp2a4 and Cyp2a5 genes, but we also find that these two genes and their antisense transcripts are expressed within different zones of the liver structure. Interestingly, while the differences in sex-biased expression of the mRNAs are established 1-2months after birth, the antisense transcripts exhibit these expression differences earlier, at 3-4weeks after birth. By analyzing published genomic data, we have identified candidate transcription factor binding sites that could account for differences in Cyp2a4/Cyp2a5 expression. Taken together, these studies characterize the first antisense RNAs within the Cyp supergene family and identify potential transcriptional and post-transcriptional mechanisms governing different Cyp2a4 and Cyp2a5 expression patterns in mouse liver.

LncRNA AFAP1-AS1 promotes proliferation ability and invasiveness of bladder cancer cells.

It was the aim of this study to explore the role and mechanism of long non-coding RNA (lncRNA) AFAP1-AS1 in the progression of bladder cancer (BCa) by in vitro experiments. AFAP1-AS1 levels in 40 pairs of clinical BCa tissue samples and normal ones collected from BCa patients were determined, and paired sample t-test was applied to compare the differences between groups. The prognosis data of patients with BCa were collected, and survival analysis and t-test were performed to specify the interplay between AFAP1-AS1 and the prognosis of BCa patients. Subsequently, AFAP1-AS1 expression level in BCa and normal cells were further confirmed by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), and Cell Counting Kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU), and transwell assays were performed to figure out the influence of this lncRNA on the proliferation ability and invasiveness of BCa cells. Meanwhile, the interaction between AFAP1-AS1 and its sense mRNA was analyzed. We used co-transfection technology to simultaneously transfect si-AFAP1-AS1 and pcDNA3.1-AFAP1 or their corresponding negative controls into BCa cells, and cell proliferation and invasion ability in different subgroups were determined to explore the underlying mechanism through which AFAP1-AS1 plays a role in BCa progression. No matter in BCa tissues or in cell samples, compared to the corresponding normal controls, AFAP1-AS1 was found highly expressed; at the same time, in invasive bladder cancer tissues, the expression level of AFAP1-AS1 was also higher than that in non-invasive tissues. Meanwhile, survival analysis revealed that patients with BCa with high expression of AFAP1-AS1 owned a shorter overall survival rate than those with low expression, indicating a negative interplay between AFAP1-AS1 expression and patients' prognosis. In addition, in BCa cell lines, according to the results of CCK-8, EDU, and transwell assays, the proliferative capacity, as well as the invasive ability of BCa cells, were found weakened after downregulation of AFAP1-AS1. Meanwhile, a negative interplay was discovered between AFAP1-AS1 and its sense mRNA. Finally, the results of cell reversal experiment using co-transfection technique revealed that overexpression of AFAP1 can reverse the inhibitory impact of lncRNAAFAP1-AS1 on the malignant ability of BCa cells. AFAP1-AS1 may enhance the proliferation ability as well as the invasiveness of BCa cells so as to aggravate the degree of BCa malignancy.

Abstract 4063: Design and development of a novel 5-FU modified siRNA against BCL2 with enhanced efficacy and the unique ability to be delivered vehicle free into cancer cells

Abstract Evasion of apoptosis is one of the major hallmarks of cancer and an important factor in chemoresistance. Thus, targeting apoptosis regulation is an important therapeutic approach. Anti-apoptotic BCL2, an oncogene and therapeutic target, is overexpressed in several different cancer types. There have been various approaches developed to target BCL2 including the FDA approved Venetoclax. In this study we have developed a modified siRNA against BCL2 that has enhanced efficacy and also can be delivered into cancer cells without any vehicle. We developed this siRNA by replacing uracil residues in the siRNA with 5-flurouracil (5-FU). This modification incorporates the effects of both 5-FU and siBCL2, to have an enhanced therapeutic effect. We tested several different modified siBCL2's, one with only the sense strand modified, one with only the antisense strand modified and one with both strands modified. We found the modified siBCL2 with both strands modified with 5-FU was most effective at inhibiting BCL2. Importantly, we were able to demonstrate via qRT-PCR and Western blot, that this modified siBCL2 retains its siRNA function and inhibits BCL2 expression in several different cancer types including colon cancer, lung cancer and lymphoma. In addition, in lymphoma cells, we found that our modified siBCL2 has better efficacy than BCL2 inhibitor Venetoclax. Finally, we were able to demonstrate that modified siBCL2 can be delivered into cancer cells without a delivery vehicle. This unique feature may be significant in overcoming the challenge of delivery, which remains a major obstacle for RNA therapeutics. This modified siBCL2 has therapeutic potential for several different cancers. Beyond BCL2, the modification approach we demonstrated may be applicable to other siRNAs and has potential as a platform technology for siRNA based therapeutics. Citation Format: Andrew Fesler, Ga-Ram Hwang, John Yuen, Jingfang Ju. Design and development of a novel 5-FU modified siRNA against BCL2 with enhanced efficacy and the unique ability to be delivered vehicle free into cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4063.

Human Cytomegalovirus Influences Host circRNA Transcriptions during Productive Infection.

Human cytomegalovirus (HCMV) is a double-strand DNA virus widely infected in human. Circular RNAs (circRNAs) are non-coding RNAs with most functions of which keep unknown, and the effects of HCMV productive infection on host circRNA transcriptions remain unclear. In this study, we profiled 283 host circRNAs that significantly altered by HCMV productive infection in human embryonic lung fibroblasts (HELF)by RNA deep sequencing and bioinformatics analysis. Among these, circSP100, circMAP3K1, circPLEKHM1, and circTRIO were validated for their transcriptions and sequences. Furthermore, characteristics of circSP100 were investigated by RT-qPCR and northern blot. It was implied that circSP100 was produced from the sense strand of the SP100 gene containing six exons. Kinetics of circSP100 and SP100 mRNA were significantly different after infection: circSP100 levels increased gradually along with infection, whereas SP100 mRNA levels increased in the beginning and dropped at 24h post-infection (hpi). Meanwhile, a total number of 257 proteins, including 10 HCMV encoding proteins, were identified potentially binding to cytoplasmic circSP100 by RNA antisense purification (RAP)and mass spectrometry. Enrichment analysis showed these proteins were mainly involved in the spliceosome, protein processing, ribosome, and phagosome pathways, suggesting multiple functions of circSP100 during HCMV infection.

Long noncoding RNA TTN-AS1 facilitates tumorigenesis and metastasis by maintaining TTN expression in skin cutaneous melanoma

The antisense transcript, emanating from the opposite strand to a protein-coding or sense strand, has been reported to have critical roles in gene regulation. The perturbation of an antisense RNA can alter the expression of sense messenger RNAs. In this study, a long noncoding RNA TTN-AS1 (lncRNA-TTN-AS1), which is transcribed in the opposite direction of the human titin (TTN) gene, has been identified and explored in skin cutaneous melanoma (SKCM). We found that the expression of TTN and lncRNA-TTN-AS1 had a significantly positive correlation in SKCM cells. Functionally, ectopic expression of TTN and lncRNA-TTN-AS1 promoted SKCM tumorigenesis and metastasis both in vitro and in vivo. Moreover, knockdown of TTN partially abrogated lncRNA-TTN-AS1 induced SKCM tumorigenesis. Mechanistically, hypomethylation of transcription initiation site was responsible for lncRNA-TTN-AS1 high expression levels. LncRNA-TTN-AS1 facilitated SKCM progression by promoting TTN expression at both transcriptional and posttranscriptional levels. As detailed, lncRNA-TTN-AS1 had a significant effect on the increase of TTN promoter activity. Besides, lncRNA-TTN-AS1 also induced the accumulation of TTN in cytoplasm by increasing the stability of TTN mRNA. Clinically, we found that high TTN and lncRNA-TTN-AS1 expression were positively correlated with poor overall survival of SKCM patients, and may be considered as novel biomarkers and drug targets for SKCM patients.

LncRNA FAM83H-AS1 promotes oesophageal squamous cell carcinoma progression via miR-10a-5p/Girdin axis.

Long non‐coding RNAs (lncRNAs) have been well demonstrated to emerge as crucial regulators in cancer progression, and they can function as regulatory network based on their interactions. Although the biological functions of FAM83H‐AS1 have been confirmed in various tumour progressions, the underlying molecular mechanisms of FAM83H‐AS1 in oesophageal squamous cell carcinoma (ESCC) remained poorly understood. To address this, we treated human oesophageal cancer cell line Eca109 cells with TGF‐β and found FAM83H‐AS1 was notably overexpressed. In the present study, FAM83H‐AS1 was observed to be significantly up‐regulated in ESCC tissues and was associated with TNM stage, pathological differentiation and lymph node metastasis. FAM83H‐AS1 reinforced oesophageal cancer cell proliferation, migration and invasion, and participated in epithelial‐to‐mesenchymal transition (EMT) process at mRNA and protein levels. In addition, a concordant regulation between FAM83H‐AS1 and its sense strand FAM83H was detected at the transcriptional and translational levels. Furthermore, FAM83H‐AS1 could act as competing endogenous RNA to affect the expression of Girdin by sponging miR‐10a‐5p verified by RIP and luciferase reporter assays. Consequently, the study provided a unique perspective of FAM83H‐AS1 in ESCC progression, which may be considered as potential biomarker and therapeutic target for ESCC therapy.

Genome-Wide Discovery of G-Quadruplexes in Wheat: Distribution and Putative Functional Roles.

G-quadruplexes are nucleic acid secondary structures formed by a stack of square planar G-quartets. G-quadruplexes were implicated in many biological functions including telomere maintenance, replication, transcription, and translation, in many species including humans and plants. For wheat, however, though it is one of the world’s most important staple food, no G-quadruplex studies have been reported to date. Here, we computationally identify putative G4 structures (G4s) in wheat genome for the first time and compare its distribution across the genome against five other genomes (human, maize, Arabidopsis, rice, and sorghum). We identified close to 1 million G4 motifs with a density of 76 G4s/Mb across the whole genome and 93 G4s/Mb over genic regions. Remarkably, G4s were enriched around three regions, two located on the antisense and one on the sense strand at the following positions: 1) the transcription start site (TSS) (antisense), 2) the first coding domain sequence (CDS) (antisense), and 3) the start codon (sense). Functional enrichment analysis revealed that the gene models containing G4 motifs within these peaks were associated with specific gene ontology (GO) terms, such as developmental process, localization, and cellular component organization or biogenesis. We investigated genes encoding MADS-box transcription factors and showed examples of G4 motifs within critical regulatory regions in the VRN-1 genes in wheat. Furthermore, comparison with other plants showed that monocots share a similar distribution of G4s, but Arabidopsis shows a unique G4 distribution. Our study shows for the first time the prevalence and possible functional roles of G4s in wheat.

Abstract IA17: Comprehensive genomic profiling of cell-free DNA enables treatment response monitoring

Abstract Liquid biopsies have advanced significantly over the past decade but are still not routinely used to guide patient care. Part of the reason is that prospective clinical trials will be needed to demonstrate clinical utility, but detection sensitivity also needs to improve. For instance, liquid biopsies are not yet capable of discerning which patients need surgery after neoadjuvant treatment or which patients need adjuvant treatment right after surgery. They are also not yet sensitive enough to determine whether it is safe to stop therapy, as BCR-ABL testing has shown for patients with CML. We reason that 1,000-fold higher detection sensitivity (10−6) will be required to provide sufficient dynamic range to guide treatment decisions in most patients. One way to improve detection sensitivity is to track more mutations per patient. This is because when tumor fraction is low in the bloodstream, only some mutations will be drawn in a blood tube, and the sampling of mutations will be random. Yet, each patient’s cancer is unique, with mutations scattered throughout the genome, and existing technologies are not well suited for tracking large numbers of distinct mutations. Conventional sequencing has an error rate of 10−3 and methods that repeatedly sequence each fragment, or model noise in normal samples, can achieve 10−4 to 10−5 but are not robust to spontaneous errors. Duplex sequencing is the most accurate method for mutation detection (10−6) but requires extremely deep sequencing to form a consensus of sense strands of each cfDNA duplex, which makes it highly inefficient and costly. In my talk, I will first describe our team’s efforts to thoroughly optimize duplex sequencing for accurate tracking of large mutation fingerprints. I will show that detection limits up to 100-fold better than ddPCR can be achieved in serial dilution series, and that this enables treatment response monitoring in patients treated for early-stage breast cancer. I will then describe a new method to improve the efficiency of duplex sequencing using mutation enrichment. I will show that it is possible to track tens of thousands of distinct mutations in cfDNA using up to 1000-fold less sequencing, and that this enables 1000-fold better detection limit than ddPCR. In all, we believe that the improved dynamic range, afforded by the higher accuracy and efficiency in cfDNA sequencing, could make treatment response monitoring feasible for more cancer patients. Citation Format: Viktor Adalsteinsson. Comprehensive genomic profiling of cell-free DNA enables treatment response monitoring [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr IA17.

Enhanced Performance of DNA Methylation Markers by Simultaneous Measurement of Sense and Antisense DNA Strands after Cytosine Conversion.

Most existing DNA methylation-based methods for detection of circulating tumor DNA (ctDNA) are based on conversion of unmethylated cytosines to uracil. After conversion, the 2 DNA strands are no longer complementary; therefore, targeting only 1 DNA strand merely utilizes half of the available input DNA. We investigated whether the sensitivity of methylation-based ctDNA detection strategies could be increased by targeting both DNA strands after bisulfite conversion. Dual-strand digital PCR assays were designed for the 3 colorectal cancer (CRC)-specific methylation markers KCNQ5, C9orf50, and CLIP4 and compared with previously reported single-strand assays. Performance was tested in tumor and leukocyte DNA, and the ability to detect ctDNA was investigated in plasma from 43 patients with CRC stages I to IV and 42 colonoscopy-confirmed healthy controls. Dual-strand assays quantified close to 100% of methylated control DNA input, whereas single-strand assays quantified approximately 50%. Furthermore, dual-strand assays showed a 2-fold increase in the number of methylated DNA copies detected when applied to DNA purified from tumor tissue and plasma from CRC patients. When the results of the 3 DNA methylation markers were combined into a ctDNA detection test and applied to plasma, the dual-strand assay format detected 86% of the cancers compared with 74% for the single-strand assay format. The specificity was 100% for both the dual- and single-strand test formats. Dual-strand assays enabled more sensitive detection of methylated ctDNA than single-strand assays.

Less Is More: Novel Hepatocyte-Targeted siRNA Conjugates for Treatment of Liver-Related Disorders

N-acetyl-galactosamine (GalNAc) conjugation enhances liver specificity for therapeutic oligonucleotides. Here we report on a novel design with improved activity and stability compared with a triantennary design. We applied a versatile monovalent serinol-GalNAc conjugation strategy. First, 1–4 serial serinol-linked GalNAc units were conjugated to terminal positions of small interfering RNA (siRNA) molecules. In primary hepatocytes, 5′ antisense GalNAc conjugates were inactive, whereas 3′ antisense and 3′ or 5′ sense conjugates displayed low activity for single GalNAc units, while 2–4 serial GalNAc conjugates were all equally potent. In mice, 5′ sense conjugates with 2–4 serial GalNAc units were all as potent as a triantennary GalNAc control (1 mg/kg). Second, increased spacing between two serial 5′ sense-conjugated GalNAc units did not affect in vitro activity. Finally, two single GalNAc units were positioned at opposite ends of the sense strand. A single dose (0.3 mg/kg) of this novel conjugate in mice showed a 3-fold reduction of serum target protein level at day 7 and 4-fold lower serum level at day 27, relative to an equimolar dose of a triantennary GalNAc conjugate of the same siRNA. Improved tritosome stability (by liquid chromatography-mass spectrometry [LC-MS] analysis) can at least partially explain the increased activity and duration of action for the novel GalNAc conjugate.

Hypermethylation-mediated down-regulation of lncRNA TBX5-AS1:2 in Tetralogy of Fallot inhibits cell proliferation by reducing TBX5 expression.

Tetralogy of Fallot (TOF) is the most common complex congenital heart disease (CHD) with uncertain cause. Although long non‐coding RNAs (lncRNAs) have been implicated in heart development and several CHDs, their role in TOF is not well understood. This study aimed to investigate how dysregulated lncRNAs contribute to TOF. Using Gene Expression Omnibus data mining, bioinformatics analysis and clinical heart tissue sample detecting, we identified a novel antisense lncRNA TBX5‐AS1:2 with unknown function that was significantly down‐regulated in injured cardiac tissues from TOF patients. LncRNA TBX5‐AS1:2 was mainly located in the nucleus of the human embryonic kidney 293 (HEK293T) cells and formed an RNA‐RNA double‐stranded structure in the overlapping region with its sense mRNA T‐box transcription factor 5 (TBX5), which is an important regulator in heart development. Knock‐down of lncRNA TBX5‐AS1:2 via promoter hypermethylation reduced TBX5 expression at both the mRNA and protein levels by affecting its mRNA stability through RNA‐RNA interaction. Moreover, lncRNA TBX5‐AS1:2 knock‐down inhibited the proliferation of HEK293T cells. In conclusion, these results indicated that lncRNA TBX5‐AS1:2 may be involved in TOF by affecting cell proliferation by targeting TBX5.

PIWI-interacting RNAs are differentially expressed during cardiac differentiation of human pluripotent stem cells.

PIWI-interacting RNAs (piRNAs) are a class of non-coding RNAs initially thought to be restricted exclusively to germline cells. In recent years, accumulating evidence has demonstrated that piRNAs are actually expressed in pluripotent, neural, cardiac and even cancer cells. However, controversy remains around the existence and function of somatic piRNAs. Using small RNA-seq samples from H9 pluripotent cells differentiated to mesoderm progenitors and cardiomyocytes we identified the expression of 447 piRNA transcripts, of which 241 were detected in pluripotency, 218 in mesoderm and 171 in cardiac cells. The majority of them originated from the sense strand of protein coding and lncRNAs genes in all stages of differentiation, though no evidences of amplification loop (ping-pong) were found. Genes hosting piRNA transcripts in cardiac samples were related to critical biological processes in the heart, like contraction and cardiac muscle development. Our results indicate that these piRNAs might have a role in fine-tuning the expression of genes involved in differentiation of pluripotent cells to cardiomyocytes.

Long noncoding RNA PAHAL modulates locust behavioural plasticity through the feedback regulation of dopamine biosynthesis.

Some long noncoding RNAs (lncRNAs) are specifically expressed in brain cells, implying their neural and behavioural functions. However, how lncRNAs contribute to neural regulatory networks governing the precise behaviour of animals is less explored. Here, we report the regulatory mechanism of the nuclear-enriched lncRNA PAHAL for dopamine biosynthesis and behavioural adjustment in migratory locusts (Locusta migratoria), a species with extreme behavioral plasticity. PAHAL is transcribed from the sense (coding) strand of the gene encoding phenylalanine hydroxylase (PAH), which is responsible for the synthesis of dopamine from phenylalanine. PAHAL positively regulates PAH expression resulting in dopamine production in the brain. In addition, PAHAL modulates locust behavioral aggregation in a population density-dependent manner. Mechanistically, PAHAL mediates PAH transcriptional activation by recruiting serine/arginine-rich splicing factor 2 (SRSF2), a transcription/splicing factor, to the PAH proximal promoter. The co-activation effect of PAHAL requires the interaction of the PAHAL/SRSF2 complex with the promoter-associated nascent RNA of PAH. Thus, the data support a model of feedback modulation of animal behavioural plasticity by an lncRNA. In this model, the lncRNA mediates neurotransmitter metabolism through orchestrating a local transcriptional loop.

Synthesis, Derivatization and Photochemical Control of ortho-Functionalized Tetrachlorinated Azobenzene-Modified siRNAs.

We report the chemical synthesis and derivatization of an ortho-functionalized tetrachlorinated azobenzene diol. A 4',4-dimethoxytrityl (DMT) phosphoramidite was synthesized for its site-specific incorporation within the sense strand of an siRNA duplex to form ortho-functionalized tetrachlorinated azobenzene-containing siRNAs (Cl-siRNAzos). Compared to a non-halogenated azobenzene, ortho-functionalized tetrachlorinated azobenzenes are capable of red-shifting the π→π* transition from the ultraviolet (UV) portion of the electromagnetic spectrum into the visible range. Within this visible range, the azobenzene molecule can be reliably converted from trans to cis with red light (660 nm), and converted back to trans with violet wavelength light (410 nm) and/or thermal relaxation. We also report the gene-silencing ability of these Cl-siRNAzos in cell culture as well as their reversible control with visible light for up to 24 hours.

Effect of 2'-5'/3'-5' phosphodiester linkage heterogeneity on RNA interference.

We report on the synthesis of siRNAs containing both 2′-5′- and 3′-5′-internucleotide linkages and their effects on siRNA structure, function, and interaction with RNAi proteins. Screening of these siRNAs against their corresponding mRNA targets showed that 2′-5′ linkages were well tolerated in the sense strand, but only at a few positions in the antisense strand. Extensive modification of the antisense strand minimally affected 5′-phosphorylation of the siRNA by kinases, however, it negatively affected siRNA loading into human AGO2. Modelling and molecular dynamics simulations were fully consistent with these findings. Furthermore, our studies indicated that the presence of a single 5′p-rN1-(2′-5′)-N2 unit in the antisense strand does not alter the ‘clover leaf’ bend and sugar puckers that are critical for anchoring the 5′-phosphate to Ago 2 MID domain. Importantly, 2′-5′-linkages had the added benefit of abrogating immune-stimulatory activity of siRNAs. Together, these results demonstrate that 2′-5′/3′-5′-modified siRNAs, when properly designed, can offer an efficient new class of siRNAs with diminished immune-stimulatory responses.

Characterization of Arabidopsis thaliana Promoter Bidirectionality and Antisense RNAs by Inactivation of Nuclear RNA Decay Pathways.

In animals, RNA polymerase II initiates transcription bidirectionally from gene promoters to produce pre-mRNAs on the forward strand and promoter upstream transcripts (PROMPTs) on the reverse strand. PROMPTs are degraded by the nuclear exosome. Previous studies based on nascent RNA approaches concluded that Arabidopsis (Arabidopsis thaliana) does not produce PROMPTs. Here, we used steady-state RNA sequencing in mutants defective in nuclear RNA decay including the exosome to reassess the existence of Arabidopsis PROMPTs. While they are rare, we identified ∼100 cases of exosome-sensitive PROMPTs in Arabidopsis. Such PROMPTs are sources of small interfering RNAs in exosome-deficient mutants, perhaps explaining why plants have evolved mechanisms to suppress PROMPTs. In addition, we found ∼200 long, unspliced and exosome-sensitive antisense RNAs that arise from transcription start sites within parts of the genome encoding 3'-untranslated regions on the sense strand. The previously characterized noncoding RNA that regulates expression of the key seed dormancy regulator, DELAY OF GERMINATION1, is a typical representative of this class of RNAs. Transcription factor genes are overrepresented among loci with exosome-sensitive antisense RNAs, suggesting a potential for widespread control of gene expression via this class of noncoding RNAs. Lastly, we assess the use of alternative promoters in Arabidopsis and compare the accuracy of existing TSS annotations.