Stem-loop Region Research Articles

5′-tRNAGly(GCC) halves generated by IRE1α are linked to the ER stress response

Transfer RNA halves (tRHs) have various biological functions. However, the biogenesis of specific 5′-tRHs under certain conditions remains unknown. Here, we report that inositol-requiring enzyme 1α (IRE1α) cleaves the anticodon stem-loop region of tRNAGly(GCC) to produce 5′-tRHs (5′-tRH-GlyGCC) with highly selective target discrimination upon endoplasmic reticulum (ER) stress. Levels of 5′-tRH-GlyGCC positively affect cancer cell proliferation and modulate mRNA isoform biogenesis both in vitro and in vivo; these effects require co-expression of two nuclear ribonucleoproteins, HNRNPM and HNRNPH2, which we identify as binding proteins of 5′-tRH-GlyGCC. In addition, under ER stress in vivo, we observe simultaneous induction of IRE1α and 5′-tRH-GlyGCC expression in mouse organs and a distantly related organism, Cryptococcus neoformans. Thus, collectively, our findings indicate an evolutionarily conserved function for IRE1α-generated 5′-tRH-GlyGCC in cellular adaptation upon ER stress.

Dimeric-molecular beacon based intramolecular strand displacement amplification enables robust analysis of miRNA

Given the critical role of miRNAs in regulating gene expression and their potential as biomarkers for various diseases, accurate and sensitive miRNA detection is essential for early diagnosis and monitoring of conditions such as cancer. In this study, we introduce a dimeric molecular beacon (Di-MB) based isothermal strand displacement amplification (ISDA) system (Di-MB-ISDA) for enhanced miRNA detection. The Di-MB system is composed of two monomeric MBs (Mono-MBs) connected by a double-stranded DNA linker with single-stranded sequences in the middle, facilitating binding with the flexible arms of the Mono-MBs. This design forms a compact, high-density structure, significantly improving biostability against nuclease degradation. In the absence of target miRNA, the Di-MB maintains its stable structure. When target miRNA is present, it binds to the stem-loop regions, causing the hairpin structure to unfold and expose the stem sequences. These sequences serve as templates for the built-in primers, triggering DNA replication through an intramolecular recognition mechanism. This spatial confinement effect accelerates the strand displacement reaction, allowing the target miRNA to initiate additional reaction cycles and amplify the detection signal. The Di-MB-ISDA system addresses key challenges such as poor biostability and limited sensitivity seen in traditional methods. By enhancing biostability and optimizing reaction conditions, this system demonstrates robust performance for miRNA detection with a detection limit of 100 pM. The findings highlight the potential of Di-MB-ISDA for sensitive and accurate miRNA analysis, paving the way for its application in biomedical study and disease diagnosis in complex biological samples.

Factor-Dependent Internal Ribosome Entry Site and -1 Programmed Frameshifting Signal in the Bemisia-Associated Dicistrovirus 2.

The dicistrovirus intergenic (IGR) IRES uses the most streamlined translation initiation mechanism: the IRES recruits ribosomes directly without using protein factors and initiates translation from a non-AUG codon. Several subtypes of dicistroviruses IRES have been identified; typically, the IRESs adopt two -to three overlapping pseudoknots with key stem-loop and unpaired regions that interact with specific domains of the ribosomal 40S and 60S subunits to direct translation. We previously predicted an atypical IGR IRES structure and a potential -1 programmed frameshift (-1 FS) signal within the genome of the whitefly Bemisia-associated dicistrovirus 2 (BaDV-2). Here, using bicistronic reporters, we demonstrate that the predicted BaDV-2 -1 FS signal can drive -1 frameshifting in vitro via a slippery sequence and a downstream stem-loop structure that would direct the translation of the viral RNA-dependent RNA polymerase. Moreover, the predicted BaDV-2 IGR can support IRES translation in vitro but does so through a mechanism that is not typical of known factorless dicistrovirus IGR IRES mechanisms. Using deletion and mutational analyses, the BaDV-2 IGR IRES is mapped within a 140-nucleotide element and initiates translation from an AUG codon. Moreover, the IRES does not bind directly to purified ribosomes and is sensitive to eIF2 and eIF4A inhibitors NSC1198983 and hippuristanol, respectively, indicating an IRES-mediated factor-dependent mechanism. Biophysical characterization suggests the BaDV-2 IGR IRES contains several stem-loops; however, mutational analysis suggests a model whereby the IRES is unstructured or adopts distinct conformations for translation initiation. In summary, we have provided evidence of the first -1 FS frameshifting signal and a novel factor-dependent IRES mechanism in this dicistrovirus family, thus highlighting the diversity of viral RNA-structure strategies to direct viral protein synthesis.

A simple, portable and multiplex LAMP-based CRISPR/Cas12a assay for visually screening genetically modified crops

With the growing controversy over the potential risk of genetically modified (GM) crops to environment and even human health worldwide, there is an urgent need to develop rapid, easy-to-operate, portable, multiplex and cost-effective methods for on-field testing of GM crops. Herein, we develop a novel multiplex loop-mediated isothermal amplification (LAMP)-based CRISPR/Cas12a method for sensitive and accurate screening of GM crops. The novel multiplex LAMP uses several pairs of stem-loop primers (SLPs) according to target genes to increase the formation ability of double stem-loop DNA to enhance LAMP efficiency. By designing the same stem-loop region of SLP pairs, multiple genes can be amplified simultaneously using universal primers to effectively avoid amplification bias. The simultaneous detection mode can increase the detection sensitivity, therefore, as low as 100 aM target genes can be simultaneously amplified and then activate CRISPR/Cas12a system to cleave reporter probe for fluorescence releasing, which can be observed under UV irradiation at an end-point manner. Further detection of real samples reveals that only 0.5% GM maize can be visually differentiated from a large amount of non-GM maize. Therefore, our method shows great promise to be applied in the testing of other GM organisms and monitoring of food authenticity under isothermal condition.

4.5SH RNA counteracts deleterious exonization of SINE B1 in mice

4.5SH RNA is a highly abundant, small rodent-specific noncoding RNA that localizes to nuclear speckles enriched in pre-mRNA-splicing regulators. To investigate the physiological functions of 4.5SH RNA, we have created mutant mice that lack the expression of 4.5SH RNA. The mutant mice exhibited embryonic lethality, suggesting that 4.5SH RNA is an essential species-specific noncoding RNA in mice. RNA-sequencing analyses revealed that 4.5SH RNA protects the transcriptome from abnormal exonizations of the antisense insertions of the retrotransposon SINE B1 (asB1), which would otherwise introduce deleterious premature stop codons or frameshift mutations. Mechanistically, 4.5SH RNA base pairs with complementary asB1-containing exons via the target recognition region and recruits effector proteins including Hnrnpm via its 5' stem loop region. The modular organization of 4.5SH RNA allows us to engineer a programmable splicing regulator to induce the skipping of target exons of interest. Our results also suggest the general existence of splicing regulatory noncoding RNAs.

An intronic RNA element modulates Factor VIII exon-16 splicing.

Pathogenic variants in the human Factor VIII (F8) gene cause Hemophilia A (HA). Here, we investigated the impact of 97 HA-causing single-nucleotide variants on the splicing of 11 exons from F8. For the majority of F8 exons, splicing was insensitive to the presence of HA-causing variants. However, splicing of several exons, including exon-16, was impacted by variants predicted to alter exonic splicing regulatory sequences. Using exon-16 as a model, we investigated the structure-function relationship of HA-causing variants on splicing. Intriguingly, RNA chemical probing analyses revealed a three-way junction structure at the 3'-end of intron-15 (TWJ-3-15) capable of sequestering the polypyrimidine tract. We discovered antisense oligonucleotides (ASOs) targeting TWJ-3-15 partially rescue splicing-deficient exon-16 variants by increasing accessibility of the polypyrimidine tract. The apical stem loop region of TWJ-3-15 also contains two hnRNPA1-dependent intronic splicing silencers (ISSs). ASOs blocking these ISSs also partially rescued splicing. When used in combination, ASOs targeting both the ISSs and the region sequestering the polypyrimidine tract, fully rescue pre-mRNA splicing of multiple HA-linked variants of exon-16. Together, our data reveal a putative RNA structure that sensitizes F8 exon-16 to aberrant splicing.

Improved discovery of RNA-binding protein binding sites in eCLIP data using DEWSeq.

Enhanced crosslinking and immunoprecipitation (eCLIP) sequencing is a method for transcriptome-wide detection of binding sites of RNA-binding proteins (RBPs). However, identified crosslink sites can deviate from experimentally established functional elements of even well-studied RBPs. Current peak-calling strategies result in low replication and high false positive rates. Here, we present the R/Bioconductor package DEWSeq that makes use of replicate information and size-matched input controls. We benchmarked DEWSeq on 107 RBPs for which both eCLIP data and RNA sequence motifs are available and were able to more than double the number of motif-containing binding regions relative to standard eCLIP processing. The improvement not only relates to the number of binding sites (3.1-fold with known motifs for RBFOX2), but also their subcellular localization (1.9-fold of mitochondrial genes for FASTKD2) and structural targets (2.2-fold increase of stem-loop regions for SLBP. On several orthogonal CLIP-seq datasets, DEWSeq recovers a larger number of motif-containing binding sites (3.3-fold). DEWSeq is a well-documented R/Bioconductor package, scalable to adequate numbers of replicates, and tends to substantially increase the proportion and total number of RBP binding sites containing biologically relevant features.

Sugarcane streak mosaic virus P1 protein inhibits unfolded protein response through direct suppression of bZIP60U splicing.

The unfolded protein response (UPR) is a cell-designated strategy that maintains the balance of protein folding in the endoplasmic reticulum (ER). UPR features a network of signal transduction pathways that reprogram the transcription, mRNA translation, and protein post-translational modification to relieve the ER stresses from unfolded/misfolded proteins. Infection with plant viruses can induce the UPR, and activated UPR often promotes plant viral infections in turn. However, the mechanism used by plant viruses to balance UPR and achieve robust infection remain largely unknown. In this study, P1SCSMV was identified as a virus-encoded RNA silencing suppressor (VSR). Heterologous overexpression of P1SCSMV via potato virus X (PVX) was found lead to programmed cell death (PCD) in Nicotiana benthamiana. Furthermore, P1SCSMV was also found to inhibit the PVX infection-triggered UPR by downregulating UPR-related genes and directly induced the distortion and collapse of the ER polygonal meshes on PVX-P1SCSMV infected N. benthamiana. Moreover, self-interaction, VSR activity, UPR inhibition, and cell death phenotype of P1SCSMV were also found to be dependent on its bipartite nuclear localization signal (NLS) (251RKRKLFPRIPLK262). P1SCSMV was found to directly bind to the stem-loop region of NbbZIP60U via its NLS and inhibit the UPR pathways, ultimately resulting in a PCD phenotype in PVX-P1SCSMV infected N. benthamiana leaves. This study also revealed the balancing role of potyviruses encoded P1SCSMV in the UPR pathway to achieve robust viral infection. This may represent a novel virulence strategy for plant viruses.

Antiviral Efficacy of RNase H-Dependent Gapmer Antisense Oligonucleotides against Japanese Encephalitis Virus.

RNase H-dependent gapmer antisense oligonucleotides (ASOs) are a promising therapeutic approach via sequence-specific binding to and degrading target RNAs. However, the efficacy and mechanism of antiviral gapmer ASOs have remained unclear. Here, we investigated the inhibitory effects of gapmer ASOs containing locked nucleic acids (LNA gapmers) on proliferating a mosquito-borne flavivirus, Japanese encephalitis virus (JEV), with high mortality. We designed several LNA gapmers targeting the 3' untranslated region of JEV genomic RNAs. In vitro screening by plaque assay using Vero cells revealed that LNA gapmers targeting a stem-loop region effectively inhibit JEV proliferation. Cell-based and RNA cleavage assays using mismatched LNA gapmers exhibited an underlying mechanism where the inhibition of viral production results from JEV RNA degradation by LNA gapmers in a sequence- and modification-dependent manner. Encouragingly, LNA gapmers potently inhibited the proliferation of five JEV strains of predominant genotypes I and III in human neuroblastoma cells without apparent cytotoxicity. Database searching showed a low possibility of off-target binding of our LNA gapmers to human RNAs. The target viral RNA sequence conservation observed here highlighted their broad-spectrum antiviral potential against different JEV genotypes/strains. This work will facilitate the development of an antiviral LNA gapmer therapy for JEV and other flavivirus infections.

The return of the "Mistigri" (virus adaptative gain by gene loss) through the SARS-CoV-2 XBB.1.5 chimera that predominated in 2023.

Severe acute respiratory syndrome coronavirus 2 XBB.1.5 is the first recombinant lineage to predominate at the country and global scales. Very interestingly, like the Marseille-4B subvariant (or B.1.160) and the pandemic variant B.1.1.7 (or Alpha) previously, it has its ORF8 gene inactivated by a stop codon. We aimed here to study the distribution of stop codons in ORF8 of XBB.1.5 and non-XBB.1.5 genomes. We identified that a stop codon was present at 89 (74%) ORF8 codons in ≥1 of 15 222 404 genomes available in GISAID. The mean proportion of genomes with a stop codon per codon was 0.11% (range, 0%-7.8%). In addition, a stop codon was detected at 15 (12%) codons in at least 1000 genomes. These 15 codons are notably located on seven stem-loop hairpin regions and in the signal peptide region for the case of the XBB.1.5 lineage (codon 8). Thus, it is very likely that stop codons in ORF8 gene contributed on at least three occasions and independently during the pandemic to the evolutionary success of a lineage that became transiently predominant. Such association of gene loss with evolutionary success, which suits the recently described Mistigri rule, is an important biological phenomenon very unknown in virology while largely described in cellular organisms.

Insights into the Differential Composition of Stem-Loop Structures of Nanoviruses and Their Impacts.

Multipartite viruses package their genomic segments independently and mainly infect plants; few of them target animals. Nanoviridae is a family of multipartite single-stranded DNA (ssDNA) plant viruses that individually encapsidate ssDNAs of ~1 kb and transmit them through aphids without replication in aphid vectors, thereby causing important diseases in host plants, mainly leguminous crops. All of these components constitute an open reading frame to perform a specific role in nanovirus infection. All segments contain conserved inverted repeat sequences, potentially forming a stem-loop structure and a conserved nonanucleotide, TAGTATTAC, within a common region. This study investigated the variations in the stem-loop structure of nanovirus segments and their impact using molecular dynamics (MD) simulations and wet lab approaches. Although the accuracy of MD simulations is limited by force field approximations and simulation time scale, explicit solvent MD simulations were successfully used to analyze the important aspects of the stem-loop structure. This study involves the mutants' design, based on the variations in the stem-loop region and construction of infectious clones, followed by their inoculation and expression analysis, based on nanosecond dynamics of the stem-loop structure. The original stem-loop structures showed more conformational stability than mutant stem-loop structures. The mutant structures were expected to alter the neck region of the stem-loop by adding and switching nucleotides. Changes in conformational stability are suggested expression variations of the stem-loop structures found in host plants with nanovirus infection. However, our results can be a starting point for further structural and functional analysis of nanovirus infection. IMPORTANCE Nanoviruses comprise multiple segments, each with a single open reading frame to perform a specific function and an intergenic region with a conserved stem-loop region. The genome expression of a nanovirus has been an intriguing area but is still poorly understood. We attempted to investigate the variations in the stem-loop structure of nanovirus segments and their impact on viral expression. Our results show that the stem-loop composition is essential in controlling the virus segments' expression level.

Synthesis, NMR kinetics and dynamic structure of a 17-mer heptaloop RNA hairpin carrying a 3-N-methyluridine nucleotide residue in the loop region

A 17-mer RNA hairpin (5'GGGAGUXAGCGGCUCCC3') carrying 3-N-methyluridine (m3U) at position X (m3U7-RNA), designed to represent the anticodon stem-loop (ACSL) region of tRNAs to study an open loop state (O-state), was synthesized, purified by HPLC, and characterized by MALDI-ToF_MS and NMR methods. 1H-NMR data revealed primary (P-state in 56.1%), secondary (S-state in 43.9%) and tertiary (∼5-6%) ACSL conformations. Exchange rate constant (kex) for interconversion between P and S states is 112 sec−1 (<Δω ∼454 rad/sec), confirming a slow exchange regime between the two states. Forward (k PS ) and backward (k SP ) rate constants are 49.166 sec−1 and 62.792 sec−1, respectively, leading to a longer life-time (20.339 msec) for the P-state and a shorter life-time (15.926 msec) for the S-state. In accordance with conformational populations determined by 1H-NMR, dynamics of the P/S/tertiary states of m3U7-RNA and its wild-type counterpart (wt-RNA) were studied by three independent MD production simulations. Cluster analysis revealed that wt-RNA reflects the structural characteristics of the ACSL region of tRNAs. The P-state of m3U7-RNA was found to be structurally similar to wt-RNA but lacks an intraloop H-bond between m3U7 and C10 (U33 and nt36 in tRNAs). In the S-state of m3U7-RNA, m3U7 flips out of the loop region. O-state loop conformations of m3U7-RNA were also clustered (4.8%), wherein the loop nucleotides m3U7.A8.G9.C10.G11 stack one after another. We propose that the O-state of m3U7-RNA is the most suitable conformation that makes the loop accessible for complementary nucleotides and for non-enzymatic primordial replication of small circular RNAs. Communicated by Ramaswamy H. Sarma

RNA Polymerase III-Dependent BoNR8 and AtR8 lncRNAs Contribute to Hypocotyl Elongation in Response to Light and Abscisic Acid.

Hypocotyl elongation is inhibited by light and promoted by darkness. The plant hormone abscisic acid (ABA) also inhibits hypocotyl elongation. However, details of the molecular mechanism that regulates the integrated effects of light and ABA signaling on hypocotyl elongation remain unclear. Long non-coding RNAs (lncRNAs; >200 nt) do not encode proteins but play many physiological roles in organisms. Until now, only a few lncRNAs related to hypocotyl elongation have been reported. The lncRNAs BoNR8 (272 nt) and AtR8 (259 nt), both of which are transcribed by RNA polymerase III, are homologous lncRNAs that are abundantly present in cabbage and Arabidopsis, respectively. These lncRNAs shared 77% sequence identity, and their predicted RNA secondary structures were similar; the non-conserved nucleotides in both sequences were positioned mainly in the stem-loop regions of the secondary structures. A previous study showed that BoNR8 regulated seed germination along with ABA and that AtR8 may be involved in innate immune function in Arabidopsis. Our results show that the expression levels of BoNR8 and AtR8 were differentially affected by light and ABA and that overexpression (OX) of both BoNR8 and AtR8 in Arabidopsis regulated hypocotyl elongation depending on light and ABA.. The expression levels of light-related genes PHYB, COP1, HY5 and PIF4 and ABA-related genes ABI3 and ABI5 were altered in the AtR8-OX and BoNR8-OX lines, and, in an ABI3-defective mutant, hypocotyl elongation was greatly increased under dark condition with the addition of ABA. These results indicate that BoNR8 and AtR8 regulate hypocotyl elongation together with ABI3 and key downstream light signaling genes.

Exosomal miR-1304-3p promotes breast cancer progression in African Americans by activating cancer-associated adipocytes

Breast cancer displays disparities in mortality between African Americans and Caucasian Americans. However, the exact molecular mechanisms remain elusive. Here, we identify miR-1304-3p as the most upregulated microRNA in African American patients. Importantly, its expression significantly correlates with poor progression-free survival in African American patients. Ectopic expression of miR-1304 promotes tumor progression in vivo. Exosomal miR-1304-3p activates cancer-associated adipocytes that release lipids and enhance cancer cell growth. Moreover, we identify the anti-adipogenic gene GATA2 as the target of miR-1304-3p. Notably, a single nucleotide polymorphism (SNP) located in the miR-1304 stem-loop region shows a significant difference in frequencies of the G allele between African and Caucasian American groups, which promotes the maturation of miR-1304-3p. Therefore, our results reveal a mechanism of the disparity in breast cancer progression and suggest a potential utility of miR-1304-3p and the associated SNP as biomarkers for predicting the outcome of African American patients.

Aptamer-enhanced the Ag(I) ion-3,3′,5,5′-tetramethylbenzdine catalytic system as a novel colorimetric biosensor for ultrasensitive and selective detection of paraquat

A facile and simple colorimetric biosensor was first established for paraquat (PQ) detection based on the aptamer-enhanced oxidation process of 3,3′,5,5′-tetramethylbenzidine (TMB) by Ag+. The study confirmed that the interaction of PQ-15 aptamer with Ag+ accelerates the electron transfer from the aptamer-Ag+ complex to dissolved oxygen, which enhances the release of superoxide anion radicals (O2̇−) and facilitates the catalytic oxidation of the chromogenic substrate. PQ-15 aptamer will preferentially bind to PQ molecules, resulting in no further enhancement of the catalytic activity of Ag+. Molecular docking results revealed that the PQ molecules are attached to the stem-loop region of the PQ-15 aptamer through σ-π conjugation interactions. The proposed method is simple that only contains Ag+ and corresponding aptamer. The limit of detection (LOD) of the constructed colorimetric biosensor for PQ detection was determined to be 16.5 μg·L-1, belowing the maximum residue limit in fruits and vegetables set by the EU. Moreover, the colorimetric biosensor showed excellent selectivity and anti-interference properties, which was validated for detecting PQ residues in several typical agricultural and water samples.

Transcriptome-wide mapping reveals a diverse dihydrouridine landscape including mRNA.

Dihydrouridine is a modified nucleotide universally present in tRNAs, but the complete dihydrouridine landscape is unknown in any organism. We introduce dihydrouridine sequencing (D-seq) for transcriptome-wide mapping of D with single-nucleotide resolution and use it to uncover novel classes of dihydrouridine-containing RNA in yeast which include mRNA and small nucleolar RNA (snoRNA). The novel D sites are concentrated in conserved stem-loop regions consistent with a role for D in folding many functional RNA structures. We demonstrate dihydrouridine synthase (DUS)-dependent changes in splicing of a D-containing pre-mRNA in cells and show that D-modified mRNAs can be efficiently translated by eukaryotic ribosomes in vitro. This work establishes D as a new functional component of the mRNA epitranscriptome and paves the way for identifying the RNA targets of multiple DUS enzymes that are dysregulated in human disease.

The Role and Targets of the RNA-Binding Protein ProQ in the Gram-Negative Bacterial Pathogen Pasteurella multocida.

The Gram-negative pathogen Pasteurella multocida is the causative agent of many important animal diseases. While a number of P. multocida virulence factors have been identified, very little is known about how gene expression and protein production is regulated in this organism. One mechanism by which bacteria regulate transcript abundance and protein production is riboregulation, which involves the interaction of a small RNA (sRNA) with a target mRNA to alter transcript stability and/or translational efficiency. This interaction often requires stabilization by an RNA-binding protein such as ProQ or Hfq. In Escherichia coli and a small number of other species, ProQ has been shown to play a critical role in stabilizing sRNA-mRNA interactions and preferentially binds to the 3' stem-loop regions of the mRNA transcripts, characteristic of intrinsic transcriptional terminators. The aim of this study was to determine the role of ProQ in regulating P. multocida transcript abundance and identify the RNA targets to which it binds. We assessed differentially expressed transcripts in a proQ mutant and identified sites of direct ProQ-RNA interaction using in vivo UV-cross-linking and analysis of cDNA (CRAC). These analyses demonstrated that ProQ binds to, and stabilizes, ProQ-dependent sRNAs and transfer RNAs in P. multocida via adenosine-enriched, highly structured sequences. The binding of ProQ to two RNA molecules was characterized, and these analyses showed that ProQ bound within the coding sequence of the transcript PmVP161_1121, encoding an uncharacterized protein, and within the 3' region of the putative sRNA Prrc13. IMPORTANCE Regulation in P. multocida involving the RNA-binding protein Hfq is required for hyaluronic acid capsule production and virulence. This study further expands our understanding of riboregulation by examining the role of a second RNA-binding protein, ProQ, in transcript regulation and abundance in P. multocida.

Deciphering the Design Rules of Toehold-Gated sgRNA for Conditional Activation of Gene Expression and Protein Degradation in Mammalian Cells.

A new class of toehold-gated gRNAs (thgRNAs) has been created to provide conditional gene regulation via RNA-mediated activation. However, the detailed design principles remain elusive. Here, we presented an investigation into the design rules for conditional gRNAs by systematically varying the toehold, stem, and flexible loop regions of thgRNA for optimal gene activation in HeLa cells. We determined that nonspecific interactions between the toehold region and the flexible loop are the main driver for the background leak observed in the OFF state. By trimming the toehold length from 15 to 5 nt, the improved thgNT-F design led to a 38-fold increase in the activated ON state with no observable background leak. The same design rule was successfully adapted to target two different regions on the mCherry mRNA with the same impressive fold change. Using the thgRNA to direct conditional protein degradation, we showed up to 8-fold knockdown of a reporter protein through activating expression of a bifunctional ubiquibody GS2-IpaH9.8. This new strategy may find many new applications for cell culture control or cell therapy by removing unwanted proteins in an RNA-responsive manner.

ASSESSMENT OF THE POSSIBILITY OF USING THE CHLOROPLAST GENOME REGION psbA-trnH FOR THE STUDY OF GENETIC POLYMORPHISM OF UKRAINIAN POPULATIONS OF MUSCARI BOTRYOIDES (L.) MILL.

The genus Muscari is represented in Ukraine by three species. One of them is M. botryoides, listed in the Red DataBook of Ukraine. M. botryoides is a morphologically polymorphic species that has been trying to be divided into several separate species for a long time. One of the arguments in favor of this division is the karyological differences between populations from different parts of the range. In particular, in the Transcarpathian region of Ukraine, dip-loid populations were found in contrast to the typical tetraploid ones for this species. However, molecular genetic studies are needed to confirm genetic polymorphism. In this work, we studied the possibility of using the chloroplast genome region psbA-trnH for DNA barcoding of Ukrainian populations of M. botryoides. We amplified and se-quenced the psbA-trnH for M. botryoides accessions from three different geographically distant Ukrainian popula-tions and compared them with the psbA-trnH sequences available in the GenBank database for M. botryoides acces-sions from Southwestern Europe and M. armeniacum. Twelve nucleotide substitutions at the beginning of the spacer were shown to be a result of a single evolutionary event, the inversion of the region corresponding to the stem-loop region in the 3΄ UTR of psbA mRNA. The unification of the position of this inversion leads to a significant change in the similarity index. In addition to this insertion, the analyzed psbA-trnH sequences also differ by one SNP and two indels of 1 bp and 7 bp in length. The inversion and the 7-bp indel are polymorphic at the intraspecific level in M. botryoides, which limits their phylogenetic use, but may be useful for microevolutionary analysis. Thus, all three ac-cessions of M. botryoides from three different regions of Western Ukraine differ in the combination of these charac-ters. These data allow us to talk about the genetic polymorphism of Ukrainian populations of M. botryoides.

ORGANIZATION AND POLYMORPHISM OF THE CHLOROPLAST GENOME REGION psbA-trnH IN REPRESENTATIVES OF THE GONIOLIMON BOISS.

Four out of twenty-two species of the genus Goniolimon are common in Ukraine: G. besserianum, G. graminifoli-um, G. rubellum and G. tataricum. Two of them, G. graminifolium and G. rubellum, are listed as vulnerable species in the Red Data Book of Ukraine. Some populations of both species are sometimes reported under different species names, manly as G. desertorum (Trautv.) Klokov. and G. rubellum (S.G. Gmel.) Klokov. To clarify these taxonomic questions, the application of molecular phylogenetic methods is necessary. Recently, the fragment between the psbA and trnH genes has been particularly actively used for similar purposes. In this work, we tested the use of the psbA-trnH chloroplast genomic region for phylogenetic analysis and DNA barcoding of representatives of the genus Goni-olimon. We amplified and sequenced the psbA-trnH sequences for samples of all four species of this genus distributed in Ukraine and compared them with the psbA-trnH sequences available in the GenBank database for species of Goni-olimon and related genera. The comparison of the available sequences showed that the representatives of the genus Goniolimon differ in twelve variable nucleotide positions, seven of which are parsimony-informative. They also differ in one oligonucleotide indel and a sequence inversion corresponding to the stem-loop region in the 3΄ UTR region of psbA mRNA. The difference between the psbA-trnH regions of Goniolimon species and representatives of related gene-ra was much higher. In addition to numerous SNPs, they also differ by a polynucleotide indel. Such significant differ-ences indicate a higher rate of evolution of the psbA-trnH region during the divergence of the genus Goniolimon compared to other regions of the chloroplast genome. On the phylogenetic dendrogram based on the comparison of psbA-trnH sequences, the Goniolimon species from Central Asia form a distinct group, which is probably due to chlo-roplast genome transfer as a result of hybridization in a common distribution area. In general, the high variability of the psbA-trnH regions in representatives of the genus Goniolimon found in our study allows us to use this region to clarify the position of controversial taxa in the group.