Decryption of sequence, structure, and functional features of SINE repeat elements in SINEUP non-coding RNA-mediated post-transcriptional gene regulation.

RNA structure folding largely influences RNA regulation by providing flexibility and functional diversity. In silico and in vitro analyses are limited in their ability to capture the intricate relationships between dynamic RNA structure and RNA functional diversity present in the cell. Here, we investigate sequence, structure and functional features of mouse and human SINE-transcribed retrotransposons embedded in SINEUPs long non-coding RNAs, which positively regulate target gene expression post-transcriptionally. In-cell secondary structure probing reveals that functional SINEs-derived RNAs contain conserved short structure motifs essential for SINEUP-induced translation enhancement. We show that SINE RNA structure dynamically changes between the nucleus and cytoplasm and is associated with compartment-specific binding to RBP and related functions. Moreover, RNA-RNA interaction analysis shows that the SINE-derived RNAs interact directly with ribosomal RNAs, suggesting a mechanism of translation regulation. We further predict the architecture of 18 SINE RNAs in three dimensions guided by experimental secondary structure data. Overall, we demonstrate that the conservation of short key features involved in interactions with RBPs and ribosomal RNA drives the convergent function of evolutionarily distant SINE-transcribed RNAs.

The status of the human gene catalogue.

Scientists have been trying to identify every gene in the human genome since the initial draft was published in 2001. In the years since, much progress has been made in identifying protein-coding genes, currently estimated to number fewer than 20,000, with an ever-expanding number of distinct protein-coding isoforms. Here we review the status of the human gene catalogue and the efforts to complete it in recent years. Beside the ongoing annotation of protein-coding genes, their isoforms and pseudogenes, the invention of high-throughput RNA sequencing and other technological breakthroughs have led to a rapid growth in the number of reported non-coding RNA genes. For most of these non-coding RNAs, the functional relevance is currently unclear; we look at recent advances that offer paths forward to identifying their functions and towards eventually completing the human gene catalogue. Finally, we examine the need for a universal annotation standard that includes all medically significant genes and maintains their relationships with different reference genomes for the use of the human gene catalogue in clinical settings.

CapTrap-Seq: A platform-agnostic and quantitative approach for high-fidelity full-length RNA transcript sequencing.

Long-read RNA sequencing is essential to produce accurate and exhaustive annotation of eukaryotic genomes. Despite advancements in throughput and accuracy, achieving reliable end-to-end identification of RNA transcripts remains a challenge for long-read sequencing methods. To address this limitation, we developed CapTrap-seq, a cDNA library preparation method, which combines the Cap-trapping strategy with oligo(dT) priming to detect 5'capped, full-length transcripts, together with the data processing pipeline LyRic. We benchmarked CapTrap-seq and other popular RNA-seq library preparation protocols in a number of human tissues using both ONT and PacBio sequencing. To assess the accuracy of the transcript models produced, we introduced a capping strategy for synthetic RNA spike-in sequences that mimics the natural 5'cap formation in RNA spike-in molecules. We found that the vast majority (up to 90%) of transcript models that LyRic derives from CapTrap-seq reads are full-length. This makes it possible to produce highly accurate annotations with minimal human intervention.

The status of the human gene catalogue.

Scientists have been trying to identify all of the genes in the human genome since the initial draft of the genome was published in 2001. Over the intervening years, much progress has been made in identifying protein-coding genes, and the estimated number has shrunk to fewer than 20,000, although the number of distinct protein-coding isoforms has expanded dramatically. The invention of high-throughput RNA sequencing and other technological breakthroughs have led to an explosion in the number of reported non-coding RNA genes, although most of them do not yet have any known function. A combination of recent advances offers a path forward to identifying these functions and towards eventually completing the human gene catalogue. However, much work remains to be done before we have a universal annotation standard that includes all medically significant genes, maintains their relationships with different reference genomes, and describes clinically relevant genetic variants.

Deep sequencing of short capped RNAs reveals novel families of noncoding RNAs.

In eukaryotes, capped RNAs include long transcripts such as messenger RNAs and long noncoding RNAs, as well as shorter transcripts such as spliceosomal RNAs, small nucleolar RNAs, and enhancer RNAs. Long capped transcripts can be profiled using cap analysis gene expression (CAGE) sequencing and other methods. Here, we describe a sequencing library preparation protocol for short capped RNAs, apply it to a differentiation time course of the human cell line THP-1, and systematically compare the landscape of short capped RNAs to that of long capped RNAs. Transcription initiation peaks associated with genes in the sense direction have a strong preference to produce either long or short capped RNAs, with one out of six peaks detected in the short capped RNA libraries only. Gene-associated short capped RNAs have highly specific 3' ends, typically overlapping splice sites. Enhancers also preferentially generate either short or long capped RNAs, with 10% of enhancers observed in the short capped RNA libraries only. Enhancers producing either short or long capped RNAs show enrichment for GWAS-associated disease SNPs. We conclude that deep sequencing of short capped RNAs reveals new families of noncoding RNAs and elucidates the diversity of transcripts generated at known and novel promoters and enhancers.

SINEUPs: a novel toolbox for RNA therapeutics.

RNA molecules have emerged as a new class of promising therapeutics to expand the range of druggable targets in the genome. In addition to 'canonical' protein-coding mRNAs, the emerging richness of sense and antisense long non-coding RNAs (lncRNAs) provides a new reservoir of molecular tools for RNA-based drugs. LncRNAs are composed of modular structural domains with specific activities involving the recruitment of protein cofactors or directly interacting with nucleic acids. A single therapeutic RNA transcript can then be assembled combining domains with defined secondary structures and functions, and antisense sequences specific for the RNA/DNA target of interest. As the first representative molecules of this new pharmacology, we have identified SINEUPs, a new functional class of natural antisense lncRNAs that increase the translation of partially overlapping mRNAs. Their activity is based on the combination of two domains: an embedded mouse inverted SINEB2 element that enhances mRNA translation (effector domain) and an overlapping antisense region that provides specificity for the target sense transcript (binding domain). By genetic engineering, synthetic SINEUPs can potentially target any mRNA of interest increasing translation and therefore the endogenous level of the encoded protein. In this review, we describe the state-of-the-art knowledge of SINEUPs and discuss recent publications showing their potential application in diseases where a physiological increase of endogenous protein expression can be therapeutic.

Low Quantity Single Strand CAGE (LQ-ssCAGE) Maps Regulatory Enhancers and Promoters.

The Cap Analysis of Gene Expression (CAGE) is a powerful method to identify Transcription Start Sites (TSSs) of capped RNAs while simultaneously measuring transcripts expression level. CAGE allows mapping at single nucleotide resolution at all active promoters and enhancers. Large CAGE datasets have been produced over the years from individual laboratories and consortia, including the Encyclopedia of DNA Elements (ENCODE) and Functional Annotation of the Mammalian Genome (FANTOM) consortia. These datasets constitute open resource for TSS annotations and gene expression analysis. Here, we provide an experimental protocol for the most recent CAGE method called Low Quantity (LQ) single strand (ss) CAGE "LQ-ssCAGE", which enables cost-effective profiling of low quantity RNA samples. LQ-ssCAGE is especially useful for samples derived from cells cultured in small volumes, cellular compartments such as nuclear RNAs or for samples from developmental stages. We demonstrate the reproducibility and effectiveness of the method by constructing 240 LQ-ssCAGE libraries from 50 ng of THP-1 cell extracted RNAs and discover lowly expressed novel enhancer and promoter-derived lncRNAs.

Antisense RNAs during early vertebrate development are divided in groups with distinct features.

Long noncoding RNAs or lncRNAs are a class of non-protein-coding RNAs that are >200 nt in length. Almost 50% of lncRNAs during zebrafish development are transcribed in an antisense direction to a protein-coding gene. However, the role of these natural antisense transcripts (NATs) during development remains enigmatic. To understand NATs in early vertebrate development, we took a computational biology approach and analyzed existing as well as novel data sets. Our analysis indicates that zebrafish NATs can be divided into two major classes based on their coexpression patterns with respect to the overlapping protein-coding genes. Group 1 NATs have characteristics similar to maternally deposited RNAs in that their levels decrease as development progresses. Group 1 NAT levels are negatively correlated with that of overlapping sense-strand protein-coding genes. Conversely, Group 2 NATs are coexpressed with overlapping protein-coding genes. In contrast to Group 1, which is enriched in genes involved in developmental pathways, Group 2 protein-coding genes are enriched in housekeeping functions. Group 1 NATs also show larger overlap and higher complementarity with the sense-strand mRNAs compared to other NATs. In addition, our transcriptomics data, quantifying RNA levels from cytoplasmic and nuclear compartments, indicates that Group 1 NATs are more abundant in the cytosol. Based on their expression pattern, cytosolic nature, and their higher complementarity to the overlapping developmental mRNAs, we speculate that Group 1 NATs function post-transcriptionally to silence spurious expression of developmental genes.

SINEUP long non-coding RNA acts via PTBP1 and HNRNPK to promote translational initiation assemblies.

SINEUPs are long non-coding RNAs (lncRNAs) that contain a SINE element, and which up-regulate the translation of target mRNA. They have been studied in a wide range of applications, as both biological and therapeutic tools, although the underpinning molecular mechanism is unclear. Here, we focused on the sub-cellular distribution of target mRNAs and SINEUP RNAs, performing co-transfection of expression vectors for these transcripts into human embryonic kidney cells (HEK293T/17), to investigate the network of translational regulation. The results showed that co-localization of target mRNAs and SINEUP RNAs in the cytoplasm was a key phenomenon. We identified PTBP1 and HNRNPK as essential RNA binding proteins. These proteins contributed to SINEUP RNA sub-cellular distribution and to assembly of translational initiation complexes, leading to enhanced target mRNA translation. These findings will promote a better understanding of the mechanisms employed by regulatory RNAs implicated in efficient protein translation.

Synthetic in vitro transcribed lncRNAs (SINEUPs) with chemical modifications enhance target mRNA translation.

Chemically modified mRNAs are extensively studied with a view toward their clinical application. In particular, long noncoding RNAs (lncRNAs) containing SINE elements, which enhance the translation of their target mRNAs (i.e., SINEUPs), have potential as RNA therapies for various diseases, such as haploinsufficiencies. To establish a SINEUP-based system for efficient protein expression, we directly transfected chemically modified in vitro transcribed (mIVT) SINEUP RNAs to examine their effects on target mRNA translation. mIVT SINEUP RNAs enhanced translation of EGFP mRNA and endogenous target Sox9 mRNA in both cultured cells and a cell-free translation system. Our findings reveal the functional role of RNA modifications in SINEUPs and suggest several broad clinical applications of such an RNA regulatory system.

An NMR-based approach reveals the core structure of the functional domain of SINEUP lncRNAs.

Long non-coding RNAs (lncRNAs) are attracting widespread attention for their emerging regulatory, transcriptional, epigenetic, structural and various other functions. Comprehensive transcriptome analysis has revealed that retrotransposon elements (REs) are transcribed and enriched in lncRNA sequences. However, the functions of lncRNAs and the molecular roles of the embedded REs are largely unknown. The secondary and tertiary structures of lncRNAs and their embedded REs are likely to have essential functional roles, but experimental determination and reliable computational prediction of large RNA structures have been extremely challenging. We report here the nuclear magnetic resonance (NMR)-based secondary structure determination of the 167-nt inverted short interspersed nuclear element (SINE) B2, which is embedded in antisense Uchl1 lncRNA and upregulates the translation of sense Uchl1 mRNAs. By using NMR 'fingerprints' as a sensitive probe in the domain survey, we successfully divided the full-length inverted SINE B2 into minimal units made of two discrete structured domains and one dynamic domain without altering their original structures after careful boundary adjustments. This approach allowed us to identify a structured domain in nucleotides 31-119 of the inverted SINE B2. This approach will be applicable to determining the structures of other regulatory lncRNAs.

Cell Based Assays of SINEUP Non-coding RNAs That Can Specifically Enhance mRNA Translation.

Targeted-protein enhancement is of importance not only for the study of biological processes but also for therapeutic and biotechnological applications. Here, we present a method to selectively up-regulate protein expression of desired genes in cultured cells by means of synthetic antisense non-coding RNAs known as SINEUPs. This positive control of gene expression is at the post-transcriptional level and exerted by an inverted short interspersed nuclear element (SINE) repeat at the 3' end of SINEUPs that comprises its effector domain (ED). SINEUPs can specifically bind to any protein-coding mRNA of choice through its binding domain (BD), a region designed to complement the sequence within the 5' untranslated region (5' UTR) and around the start codon of the mRNA. Target-specific SINEUPs designed in this manner are transfected to cultured cells, and protein and RNA are extracted for downstream analyses, generally 24-48 h post-transfection. SINEUP-induced protein up-regulation is detected by Western-blot analysis and RNA expression is measured using real-time quantitative reverse transcription PCR. We have observed that BD design is critical for achieving optimum SINEUP activity and that testing different BD sizes and positions with respect to the start codon of the target mRNA is recommended. Therefore, we describe here a semi-automated high-throughput imaging method based on fluorescence detection that can be implemented to target mRNA fused with green fluorescent protein (GFP). SINEUPs specifically enhance translation within normal physiological range of the cell, without altering the target transcript level. This method has been successfully employed against a range of endogenous and exogenous targets, in a wide variety of human, mouse, and insect cell lines along with in vivo systems. Moreover, SINEUPs have been reported to increase antibody production and work as an RNA therapeutic against haploinsufficient genes. The versatile and modular nature of SINEUPs makes them a suitable tool for gene-specific translational control.

Identification of functional features of synthetic SINEUPs, antisense lncRNAs that specifically enhance protein translation.

SINEUPs are antisense long noncoding RNAs, in which an embedded SINE B2 element UP-regulates translation of partially overlapping target sense mRNAs. SINEUPs contain two functional domains. First, the binding domain (BD) is located in the region antisense to the target, providing specific targeting to the overlapping mRNA. Second, the inverted SINE B2 represents the effector domain (ED) and enhances translation. To adapt SINEUP technology to a broader number of targets, we took advantage of a high-throughput, semi-automated imaging system to optimize synthetic SINEUP BD and ED design in HEK293T cell lines. Using SINEUP-GFP as a model SINEUP, we extensively screened variants of the BD to map features needed for optimal design. We found that most active SINEUPs overlap an AUG-Kozak sequence. Moreover, we report our screening of the inverted SINE B2 sequence to identify active sub-domains and map the length of the minimal active ED. Our synthetic SINEUP-GFP screening of both BDs and EDs constitutes a broad test with flexible applications to any target gene of interest.

[Significance of Allowing Pharmacy Students to Attend Visits to Home Care Patients].

In 2006, with the admission of a new batch of students, pharmaceutical education became a 6-year course. This was a result of the urgent need to train a new generation of pharmacists to respond to increasingly advanced and intricate medical care as well as the specific need to coordinate with multiple occupational categories. Meanwhile, with Japan becoming an aged society, medical care has undergone functional differentiation, and home care is now being promoted. As part of an 11- week practical course for 5th-year practical training, students attended visits to home care patients from an early stage, making it possible for them to be present at multiple visits. This was highly significant because it allowed students to experience various disease states of different patients and increase their practical knowledge of pharmaceuticals. This study explores the case example of proposals made by pharmacy students for improving medication-related problems in home care patients during 5th-year practical training.

Characterization of piRNAs across postnatal development in mouse brain.

PIWI-interacting RNAs (piRNAs) are responsible for maintaining the genome stability by silencing retrotransposons in germline tissues- where piRNAs were first discovered and thought to be restricted. Recently, novel functions were reported for piRNAs in germline and somatic cells. Using deep sequencing of small RNAs and CAGE of postnatal development of mouse brain, we identified piRNAs only in adult mouse brain. These piRNAs have similar sequence length as those of MILI-bound piRNAs. In addition, we predicted novel candidate regulators and putative targets of adult brain piRNAs.

Remodeling of retrotransposon elements during epigenetic induction of adult visual cortical plasticity by HDAC inhibitors.

BACKGROUND: The capacity for plasticity in the adult brain is limited by the anatomical traces laid down during early postnatal life. Removing certain molecular brakes, such as histone deacetylases (HDACs), has proven to be effective in recapitulating juvenile plasticity in the mature visual cortex (V1). We investigated the chromatin structure and transcriptional control by genome-wide sequencing of DNase I hypersensitive sites (DHSS) and cap analysis of gene expression (CAGE) libraries after HDAC inhibition by valproic acid (VPA) in adult V1. RESULTS: We found that VPA reliably reactivates the critical period plasticity and induces a dramatic change of chromatin organization in V1 yielding significantly greater accessibility distant from promoters, including at enhancer regions. VPA also induces nucleosome eviction specifically from retrotransposon (in particular SINE) elements. The transiently accessible SINE elements overlap with transcription factor-binding sites of the Fox family. Mapping of transcription start site activity using CAGE revealed transcription of epigenetic and neural plasticity-regulating genes following VPA treatment, which may help to re-program the genomic landscape and reactivate plasticity in the adult cortex. CONCLUSIONS: Treatment with HDAC inhibitors increases accessibility to enhancers and repetitive elements underlying brain-specific gene expression and reactivation of visual cortical plasticity.

SINEUPs are modular antisense long non-coding RNAs that increase synthesis of target proteins in cells.

Despite recent efforts in discovering novel long non-coding RNAs (lncRNAs) and unveiling their functions in a wide range of biological processes their applications as biotechnological or therapeutic tools are still at their infancy. We have recently shown that AS Uchl1, a natural lncRNA antisense to the Parkinson's disease-associated gene Ubiquitin carboxyl-terminal esterase L1 (Uchl1), is able to increase UchL1 protein synthesis at post-transcriptional level. Its activity requires two RNA elements: an embedded inverted SINEB2 sequence to increase translation and the overlapping region to target its sense mRNA. This functional organization is shared with several mouse lncRNAs antisense to protein coding genes. The potential use of AS Uchl1-derived lncRNAs as enhancers of target mRNA translation remains unexplored. Here we define AS Uchl1 as the representative member of a new functional class of natural and synthetic antisense lncRNAs that activate translation. We named this class of RNAs SINEUPs for their requirement of the inverted SINEB2 sequence to UP-regulate translation in a gene-specific manner. The overlapping region is indicated as the Binding Doman (BD) while the embedded inverted SINEB2 element is the Effector Domain (ED). By swapping BD, synthetic SINEUPs are designed targeting mRNAs of interest. SINEUPs function in an array of cell lines and can be efficiently directed toward N-terminally tagged proteins. Their biological activity is retained in a miniaturized version within the range of small RNAs length. Its modular structure was exploited to successfully design synthetic SINEUPs targeting endogenous Parkinson's disease-associated DJ-1 and proved to be active in different neuronal cell lines. In summary, SINEUPs represent the first scalable tool to increase synthesis of proteins of interest. We propose SINEUPs as reagents for molecular biology experiments, in protein manufacturing as well as in therapy of haploinsufficiencies.

Engineering mammalian cell factories with SINEUP noncoding RNAs to improve translation of secreted proteins.

Whenever the function of a recombinant protein depends on post-translational processing, mammalian cells become an indispensable tool for their production. This is particularly true for biologics and therapeutic monoclonal antibodies (MAbs). Despite some drawbacks, Chinese Hamster Ovary (CHO) cells are the workhorse for MAbs production in academia and industry. Several methodologies have been adopted to improve expression and stability, including methods based on selective pressure or cell engineering. We have previously identified SINEUPs as a new functional class of natural and synthetic long non-coding RNAs that through the activity of an inverted SINEB2 element are able to promote translation of partially overlapping sense coding mRNAs. Here we show that by taking advantage of their modular structure, synthetic SINEUPs can be designed to increase production of secreted proteins. Furthermore, by experimentally validating antisense to elastin (AS-eln) RNA as a natural SINEUP, we show that SINEUP-mediated control may target extracellular proteins. These results lead us to propose synthetic SINEUPs as new versatile tools to optimize production of secreted proteins in manufacturing pipelines and natural SINEUPs as new regulatory RNAs in the secretory pathways.

Widespread genome transcription: new possibilities for RNA therapies.

Comprehensive analysis of mammalian transcriptomes has surprisingly revealed that a major fraction of the RNAs produced by mammalian cells and tissues is comprised of long non-coding RNAs (lncRNAs). Such RNAs were previously disregarded as useless, but recent functional studies have revealed that they have multiple regulatory functions. A large subset of these lncRNAs are antisense to protein-coding genes; such RNAs are particularly attractive to researchers because their functions are better understood than other lncRNAs and their action can be easily modulated and engineered by modifying the antisense region. We discuss various aspects of regulation by antisense RNAs and other small nucleic acids and the challenges to bring these technologies to gene therapy. Despite several remaining issues related to delivery, RNA stability, side effects, and toxicity, the field is moving quickly towards future biotechnological and health applications. Therapies based on lncRNAs may be the key to increased cell-specificity of future gene therapies.

His166 is the Schiff base proton acceptor in attractant phototaxis receptor sensory rhodopsin I.

Photoactivation of attractant phototaxis receptor sensory rhodopsin I (SRI) in Halobacterium salinarum entails transfer of a proton from the retinylidene chromophore's Schiff base (SB) to an unidentified acceptor residue on the cytoplasmic half-channel, in sharp contrast to other microbial rhodopsins, including the closely related repellent phototaxis receptor SRII and the outward proton pump bacteriorhodopsin, in which the SB proton acceptor is an aspartate residue salt-bridged to the SB in the extracellular (EC) half-channel. His166 on the cytoplasmic side of the SB in SRI has been implicated in the SB proton transfer reaction by mutation studies, and mutants of His166 result in an inverted SB proton release to the EC as well as inversion of the protein's normally attractant phototaxis signal to repellent. Here we found by difference Fourier transform infrared spectroscopy the appearance of Fermi-resonant X-H stretch modes in light-minus-dark difference spectra; their assignment with (15)N labeling and site-directed mutagenesis demonstrates that His166 is the SB proton acceptor during the photochemical reaction cycle of the wild-type SRI-HtrI complex.