Reyad A. Elbarbary

Retrotransposons as regulators of gene expression

Parasitic DNAs help and hinder evolution Transposable elements are parasitic DNAs that can duplicate themselves and jump around their host genomes. They can both disrupt gene function and drive genome evolution. Elbarbary et al. review the roles of two classes of transposable elements in gene regulation and disease: long interspersed elements (LINEs) and short interspersed elements (SINEs). Roughly a third of the human genome consists of LINEs and SINEs. They contribute to a broad range of important genome and gene regulatory features, while at the same time being responsible for number of human diseases. Science, this issue p. 10.1126/science.aac7247 BACKGROUND Genomes are subject to two types of changes: changes to the DNA sequence, and changes that are epigenetic in nature. Changes to the DNA sequence can result from errors made during DNA replication and/or repair, or from the insertion of mobile DNA. Mobile DNAs, also called transposable elements (TEs), have the potential to provide regulatory and/or protein-coding sequences at a new integration site. Depending on its nucleotide sequence and genomic insertion site, an individual TE can disrupt gene expression, directly or indirectly create an advantageous modification to gene expression, or be of no immediate consequence. Changes can be genetic, epigenetic, or both. In this way, TEs are molecular parasites and evolutionary drivers, each role originating from the ability to insert into, spread through, and restructure genomes. This review focuses on the types of TEs that transpose via RNA intermediates—retrotransposons—that are not bounded by long terminal repeats. The most abundant retrotransposons in animal genomes come in two forms: long interspersed elements (LINEs) and short interspersed elements (SINEs). ADVANCES The advent of deep genomic and transcriptomic sequencing, together with studies of individual LINE or SINE functions, has led to a greater appreciation of how the two TEs influence gene expression. Our review focuses principally on data that derive from human and mouse studies. These data demonstrate that LINEs and SINEs perform many diverse roles within cells. As DNA sequences, they can regulate gene transcription by altering chromatin structure and by functioning as enhancers or promoters. When transcribed as part of a larger transcript, they can create new transcript isoforms (by influencing alternative pre-mRNA splicing or 3′-end formation), alter mRNA localization, change mRNA stability, tune the level of mRNA translation, or encode amino acids that diversify the proteome. Further, the RNA transcripts of LINEs or SINEs may themselves function to regulate gene expression. Through their various roles, TEs influence many aspects of cellular metabolism, including the ability to divide, migrate, differentiate, and respond to stress. OUTLOOK TEs continue to spread throughout our genomes in both gametes and somatic tissues, introducing new gene regulatory activities or causing disease. Although deep transcriptome sequencing has identified a myriad of SINE-containing noncoding RNAs, the functional importance of most of these transcripts remains unknown. Additionally, we need to understand the consequence of the very high degree of TE transposition in our brains. We also need to uncover the determinants that influence the effect of a specific SINE on gene expression, and, given that different organisms can contain SINEs of distinct origins, the extent to which these SINEs contribute to species-specific differences. Moreover, it will be important to determine the extent to which independently evolved SINEs have been co-opted for similar functions. Some of the steps in the expression of mammalian genes that can be affected by cis- or trans-acting LINEs or SINEs. LINE and SINE genomic insertions can regulate gene expression by altering transcription and/or chromatin structure. When embedded in the transcripts of RNA polymerase II (Pol II)–transcribed genes, SINEs can influence nuclear pre-mRNA splicing, nuclear mRNA retention in paraspeckles, cytoplasmic mRNA stability, or cytoplasmic mRNA translation. ARE-BP, AU-rich element–binding protein. ILLUSTRATION: K.SUTLIFF/SCIENCE Transposable elements (TEs) are both a boon and a bane to eukaryotic organisms, depending on where they integrate into the genome and how their sequences function once integrated. We focus on two types of TEs: long interspersed elements (LINEs) and short interspersed elements (SINEs). LINEs and SINEs are retrotransposons; that is, they transpose via an RNA intermediate. We discuss how LINEs and SINEs have expanded in eukaryotic genomes and contribute to genome evolution. An emerging body of evidence indicates that LINEs and SINEs function to regulate gene expression by affecting chromatin structure, gene transcription, pre-mRNA processing, or aspects of mRNA metabolism. We also describe how adenosine-to-inosine editing influences SINE function and how ongoing retrotransposition is countered by the body’s defense mechanisms.

Modulation of Gene Expression by Human Cytosolic tRNase ZL through 5′-Half-tRNA

A long form (tRNase ZL) of tRNA 3′ processing endoribonuclease (tRNase Z, or 3′ tRNase) can cleave any target RNA at any desired site under the direction of artificial small guide RNA (sgRNA) that mimics a 5′-half portion of tRNA. Based on this enzymatic property, a gene silencing technology has been developed, in which a specific mRNA level can be downregulated by introducing into cells a synthetic 5′-half-tRNA that is designed to form a pre-tRNA-like complex with a part of the mRNA. Recently 5′-half-tRNA fragments have been reported to exist stably in various types of cells, although little is know about their physiological roles. We were curious to know if endogenous 5′-half-tRNA works as sgRNA for tRNase ZL in the cells. Here we show that human cytosolic tRNase ZL modulates gene expression through 5′-half-tRNA. We found that 5′-half-tRNAGlu, which co-immunoprecipitates with tRNase ZL, exists predominantly in the cytoplasm, functions as sgRNA in vitro, and downregulates the level of a luciferase mRNA containing its target sequence in human kidney 293 cells. We also demonstrated that the PPM1F mRNA is one of the genuine targets of tRNase ZL guided by 5′-half-tRNAGlu. Furthermore, the DNA microarray data suggested that tRNase ZL is likely to be involved in the p53 signaling pathway and apoptosis.

Human cytosolic tRNase ZL can downregulate gene expression through miRNA

A long form of tRNase Z (tRNase ZL) can cleave any target RNA at any desired site under the direction of artificial small guide RNA including ∼25‐nucleotide hook‐shaped RNA. Here we show that human miR‐103 can form a hook structure to guide target RNA cleavage by human cytosolic tRNase ZL in vitro. In vivo analyses using luciferase mRNAs modified to contain miR‐103 target sequences in their 3′ untranslated regions indicated that miR‐103 downregulates gene expression through directing mRNA cleavage by tRNase ZL. The present data suggest the possibility that human cytosolic tRNase ZL modulates gene expression through a subset of microRNAs in the cells.

Inhibition of vascular endothelial growth factor expression by TRUE gene silencing

Pathogenic angiogenesis in various diseases including cancer, autoimmune diseases, and age-related macular degeneration is thought to be regressed with anti-angiogenic drugs. TRUE gene silencing is a new technology to eliminate a specific mRNA using synthetic sgRNA and cellular tRNase Z(L). To discover anti-angiogenic sgRNAs, we applied TRUE silencing to the VEGF gene. We examined eight sgRNAs for efficacy in targeting exogenous human VEGF mRNA. Many of them worked efficiently in 293 and HeLa cells. Two of them downregulated the endogenous VEGF gene expression in HeLa cells very efficiently, and the efficacy of these two sgRNAs surpassed that of siRNA extremely.

A naked RNA heptamer targeting the human Bcl-2 mRNA induces apoptosis of HL60 leukemia cells

tRNase Z(L)-utilizing efficacious gene silencing is a gene control technology, which is based on the property that tRNase Z(L) can cleave any target RNA under the direction of an appropriate small guide RNA (sgRNA). To find therapeutic sgRNAs to cure hematological malignancies, we investigated behavior of heptamer-type sgRNA. We demonstrated that a heptamer, mh1(Bcl-2), which targets the human Bcl-2 mRNA, can be taken up by cells without any transfection reagents and that it can induce apoptosis of the leukemia cells. Mouse xenograft experiments showed that a median survival of the mh1(Bcl-2)-treated mice was longer than that of the control mice.

Tudor-SN–mediated endonucleolytic decay of human cell microRNAs promotes G1/S phase transition

Breaking down miRNAs Although much work has examined microRNA (miRNA) biogenesis, relatively little is known about miRNA decay. Elbarbary et al. now identify Tudor-SN, an endonuclease that interacts with the RNA-induced silencing complex. Tudor-SN targets miRNAs at CA and UA dinucleotides located more than five nucleotides from miRNA ends. Tudor-SN-mediated miRNA decay removes miRNAs that silence genes encoding proteins that are critical for the G1-to-S phase transition in the cell cycle. Science, this issue p. 859 An endonuclease initiates decay of microRNAs that regulate the cell cycle. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression. The pathways that mediate mature miRNA decay are less well understood than those that mediate miRNA biogenesis. We found that functional miRNAs are degraded in human cells by the endonuclease Tudor-SN (TSN). In vitro, recombinant TSN initiated the decay of both protein-free and Argonaute 2–loaded miRNAs via endonucleolytic cleavage at CA and UA dinucleotides, preferentially at scissile bonds located more than five nucleotides away from miRNA ends. Cellular targets of TSN-mediated decay defined using microRNA sequencing followed this rule. Inhibiting TSN-mediated miRNA decay by CRISPR-Cas9 knockout of TSN inhibited cell cycle progression by up-regulating a cohort of miRNAs that down-regulates mRNAs that encode proteins critical for the G1-to-S phase transition. Our study indicates that targeting TSN nuclease activity could inhibit pathological cell proliferation.

Elimination of Specific miRNAs by Naked 14-nt sgRNAs

tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing) is a newly developed technology to suppress mammalian gene expression. TRUE gene silencing works on the basis of a unique enzymatic property of mammalian tRNase ZL, which is that it can recognize a pre-tRNA-like or micro-pre-tRNA-like complex formed between target RNA and artificial small guide RNA (sgRNA) and can cleave any target RNA at any desired site. There are four types of sgRNA, 5′-half-tRNA, RNA heptamer, hook RNA, and ∼14-nt linear RNA. Here we show that a 14-nt linear-type sgRNA against human miR-16 can guide tRNase ZL cleavage of miR-16 in vitro and can downregulate the miR-16 level in HEK293 cells. We also demonstrate that the 14-nt sgRNA can be efficiently taken up without any transfection reagents by living cells and can exist stably in there for at least 24 hours. The naked 14-nt sgRNA significantly reduced the miR-16 level in HEK293 and HL60 cells. Three other naked 14-nt sgRNAs against miR-142-3p, miR-206, and miR-19a/b are also shown to downregulate the respective miRNA levels in various mammalian cell lines. Our observations suggest that in general we can eliminate a specific cellular miRNA at least by ∼50% by using a naked 14-nt sgRNA on the basis of TRUE gene silencing.

UPF1 helicase promotes TSN-mediated miRNA decay

While microRNAs (miRNAs) regulate the vast majority of protein-encoding transcripts, little is known about how miRNAs themselves are degraded. We recently described Tudor-staphylococcal/micrococcal-like nuclease (TSN)-mediated miRNA decay (TumiD) as a cellular pathway in which the nuclease TSN promotes the decay of miRNAs that contain CA and/or UA dinucleotides. While TSN-mediated degradation of either protein-free or AGO2-loaded miRNAs does not require the ATP-dependent RNA helicase UPF1 in vitro, we report here that cellular TumiD requires UPF1. Results from experiments using AGO2-loaded miRNAs in duplex with target mRNAs indicate that UPF1 can dissociate miRNAs from their mRNA targets, making the miRNAs susceptible to TumiD. miR-seq (deep sequencing of miRNAs) data reveal that the degradation of ∼50% of candidate TumiD targets in T24 human urinary bladder cancer cells is augmented by UPF1. We illustrate the physiological relevance by demonstrating that UPF1-augmented TumiD promotes the invasion of T24 cells in part by degrading anti-invasive miRNAs so as to up-regulate the expression of proinvasive proteins.

Screening of a heptamer-type sgRNA library for potential therapeutic agents against hematological malignancies.

tRNase-Z(L)-utilizing efficacious (TRUE) gene silencing is an RNA-mediated gene expression control technology that has therapeutic potential. This technology is based on the property of tRNase Z(L) that it can cleave any target RNA at any desired site under the direction of an appropriate artificial small guide RNA (sgRNA). To search for novel potential therapeutic sgRNAs for hematological malignancies, we screened a library composed of 156 sgRNAs, and found that 20 sgRNAs can efficiently induce apoptosis in leukemia and/or myeloma cells. Furthermore, we demonstrated that 4 of the 20 sgRNAs can reduce growth rates of HL60 cells in mouse xenograft models.

Coupling pre-mRNA splicing and 3′ end formation to mRNA export: alternative ways to punch the nuclear export clock

How does a mammalian cell determine when newly synthesized mRNAs are fully processed and appropriate for nuclear export? Müller-McNicoll and colleagues (pp. 553-566) expand on mechanisms known to be mediated by nuclear export factor 1 (NXF1) by describing SR proteins as NXF1 adaptors that flag alternatively spliced and polyadenylated mRNA isoforms as cargo ready for the cytoplasm.