RNA Duplexes Research Articles

A naked-eye colorimetric molecular “light switch” based on ruthenium(II) polypyridyl complex [Ru(phen)2ttbd]2+ as binder and stabilizer for RNA duplex and triplex

Binding of [Ru(phen)2ttbd]2+ (phen = 1,10-phenanthroline, ttbd = 4-(6-propenylpyrido-[3,2-a]- phenzain-10-yl-benzene-1,2-diamine) to the RNA triplex poly(U-A*U) (herein “-” and “*” refer to the Watson-Crick and Hoogsteen binding, respectively) and the duplex poly(A-U) have been investigated by spectral technology and viscosity method. Analysis of spectral titrations and viscosity experiments as well as melting measurements suggest that [Ru(phen)2ttbd]2+ binds to the studied RNA triplex and duplex through intercalation, while its binding constant toward the triplex is greater than the duplex. Luminescent titrations indicate that [Ru(phen)2ttbd]2+ can act as a molecular “light switch” for the two RNAs and the switch effect can be detected by the naked-eye. Moreover, the “light switch” can be repeatedly cycled off and on by adjusting the pH of the solution, whereas color change in the case of the triplex is more significant compared with the duplex. To our knowledge, [Ru(phen)2ttbd]2+ is the first small molecule capable of serving as a pH-controlled reversible visual molecular “light switch” for both the RNA triplex poly(U-A*U) and duplex poly(A-U). Thermal denaturation experiments suggest that [Ru(phen)2ttbd]2+ can obviously increase the triplex stabilization, while it stabilizing third-strand is more marked in comparison with the template duplex of the triplex, indicating this complex preferentially binds to third-strand. The obtained results may be useful for understanding the binding of Ru(II) polypyridyl complexes to RNAs.

Phytophthora effector PSR1 hijacks the host pre-mRNA splicing machinery to modulate small RNA biogenesis and plant immunity.

Phytophthora effector PSR1 suppresses small RNA (sRNA)-mediated immunity in plants, but the underlying mechanism remains unknown. Here, we show that Phytophthora suppressor of RNA silencing 1 (PSR1) contributes to the pathogenicity of Phytophthora sojae and specifically binds to three conserved C-terminal domains of the eukaryotic PSR1-Interacting Protein 1 (PINP1). PINP1 encodes PRP16, a core pre-mRNA splicing factor that unwinds RNA duplexes and binds to primary microRNA transcripts and general RNAs. Intriguingly, PSR1 decreased both RNA helicase and RNA-binding activity of PINP1, thereby dampening sRNA biogenesis and RNA metabolism. The PSR1-PINP1 interaction caused global changes in alternative splicing (AS). A total of 5,135 genes simultaneously exhibited mis-splicing in both PSR1-overexpressing and PINP1-silenced plants. AS upregulated many mRNA transcripts that had their introns retained. The high occurrence of intron retention in AS-induced transcripts significantly promoted Phytophthora pathogen infection in Nicotiana benthamiana, and this might be caused by the production of truncated proteins. Taken together, our findings reveal a key role for PINP1 in regulating sRNA biogenesis and plant immunity.

Role of tetK-siRNA in inhibition the biofilm formation as a first line of antibiotic resistance by regulation the TetK drug efflux pump in staphylococcus aureus

The Staphylococcus aureus (S. aureus) is a common cause of different infections in the community. To determine the effect of siRNA on the mRNA of the tetK gene in S. aureus, 21-23 bp small interfering RNA (siRNA) duplexes were constructed against the mRNA of the tetK gene. The effect of siRNA on tetK mRNA expression was determined using reverse transcription PCR (RT-PCR). To assess changes in biofilm formation in response to siRNA activity, the usual tube technique was adopted. In vitro, tetK-siRNAs inhibited S. aureus mRNA expression and activity. The efficacy of siRNA was determined by comparing the biofilm formation in S. aureus before and after tetK-siRNA was introduced into the bacteria. In this investigation, both modified tetK-siRNA sequence and unmodified tetK-siRNA sequence, were employed. RT-qPCR revealed that both modified tetK-siRNA sequence and unmodified tetK-siRNA sequence significantly suppressed the expression of tetK-mRNA, P = (0.04, and 0.03) respectively at (P < 0.05) comparison with control.

Inside Cover: Enzyme‐Free Copying of 12 Bases of RNA with Dinucleotides (Angew. Chem. Int. Ed. 29/2022)

Dinucleotides copy RNA in the absence of enzymes or ribozymes. Genetic copying, producing the complementary strand to a template, drives replication. Self-replication, based solely on molecular recognition and chemical reactivity, is a mainstay of the “RNA world” hypothesis for the origin of life. Dimers, the very first products of oligomerization, are superior building blocks in dilute aqueous solution, as now shown by Clemens Richert and co-workers in their Communication (DOI: 10.1002/anie.202203067). (Illustration of RNA duplexes based on PDB entry 1qc0).

The interaction between long non-coding RNA LINC01564 and POU2F1 promotes the proliferation and metastasis of gastric cancer

BackgroundAn increasing number of studies have demonstrated that long non-coding RNAs (lncRNAs) serve as key regulators in tumor development and progression. However, only a few lncRNAs have been functionally characterized in gastric cancer (GC).MethodsBioinformatics analysis was conducted to find lncRNAs that are associated with GC metastasis. RNA FISH, RIP, and RNA pull down assays were used to study the complementary binding of LINC01564 complementary to the 3′UTR of transcription factor POU2F1. The transcription activation of LINC01564 by POU2F1 as a transcription factor was examined by ChIP assay. In vitro assays such as MTT, cell invasion assay, and clonogenic assay were conducted to examined the impacts of LINC01564 and POU2F1 on GC cell proliferation and invasion. Experiments in vivo were performed to access the impacts of LINC01564 and POU2F1 on GC metastasis.ResultsThe results showed that LINC01564 complementary bound to the 3′UTR of POU2F1 to form an RNA duplex, whereby stabilizing POU2F1 mRNA and increasing the enrichment in cells. The level of LINC01564 was also increased by POU2F1 through transcription activation. In vitro assays showed that LINC01564 promoted the proliferation, invasion and migration of GC cells through increasing POU2F1. In vivo experiments indicate the promotion of GC proliferation and metastasis by the interaction between LINC01564 and POU2F1.ConclusionTaken together, our results indicate that the interaction between LINC01564 and POU2F1 promotes the proliferation, migration and invasion of GC cells.

Binding properties of ruthenium(II) complexes [Ru(phen)2(7-R-dppz)]2+ (R = methyl or bromine) toward poly(U)•poly(A) RNA duplex

Two Ru(II) complexes containing different substituents, [Ru(phen)2(7-CH3-dppz)]2+ (Ru1) and [Ru(phen)2(7-Br-dppz)]2+ (Ru2), have been synthesized in this study. The binding properties of Ru1 and Ru2 with the duplex RNA poly(U)•poly(A) (where “•” denotes the Watson – Crick base pairing) have been researched by biophysical techniques and viscosity measurements. Analysis of spectral titrations and viscosity measurements indicate that Ru1 and Ru2 bind to the duplex via intercalative, and the binding affinity of Ru1 with the duplex is remarkably higher than that of Ru2. Furthermore, fluorescence emission spectra demonstrates that although complexes Ru1 and Ru2 can act as molecular “light switches” for the duplex RNA, alters in fluorescence emission of Ru1 and Ru2 are prominent differences, and the effectiveness of Ru1 is more remarkable compared with that of Ru2. The melting experiments suggest that the duplex RNA stabilizing effects of Ru1 and Ru2 differ from each other, among them, complex Ru1 can obviously enhance the stability of the duplex RNA, while Ru2 has only a slightly stabilizing effect for the duplex RNA, indicating that Ru1 preferentially binds to RNA duplex over Ru2. The obtained results indicate that subtle modifications of the intercalative ligand of Ru(II) polypyridyl complex with either methyl or bromide group have a significant effect on the duplex-binding discrimination.

Dimeric assembly of human Suv3 helicase promotes its RNA unwinding function in mitochondrial RNA degradosome for RNA decay.

Human Suv3 is a unique homodimeric helicase that constitutes the major component of the mitochondrial degradosome to work cooperatively with exoribonuclease PNPase for efficient RNA decay. However, the molecular mechanism of how Suv3 is assembled into a homodimer to unwind RNA remains elusive. Here, we show that dimeric Suv3 preferentially binds to and unwinds DNA–DNA, DNA–RNA, and RNA–RNA duplexes with a long 3′ overhang (≥10 nucleotides). The C‐terminal tail (CTT)‐truncated Suv3 (Suv3ΔC) becomes a monomeric protein that binds to and unwinds duplex substrates with ~six to sevenfold lower activities relative to dimeric Suv3. Only dimeric Suv3, but not monomeric Suv3ΔC, binds RNA independently of ATP or ADP, and is capable of interacting with PNPase, indicating that dimeric Suv3 assembly ensures its continuous association with RNA and PNPase during ATP hydrolysis cycles for efficient RNA degradation. We further determined the crystal structure of the apo‐form of Suv3ΔC, and SAXS structures of dimeric Suv3 and PNPase–Suv3 complex, showing that dimeric Suv3 caps on the top of PNPase via interactions with S1 domains, and forms a dumbbell‐shaped degradosome complex with PNPase. Overall, this study reveals that Suv3 is assembled into a dimeric helicase by its CTT for efficient and persistent RNA binding and unwinding to facilitate interactions with PNPase, promote RNA degradation, and maintain mitochondrial genome integrity and homeostasis.

Mechanism of reaction of RNA-dependent RNA polymerase from SARS-CoV-2

SummaryWe combine molecular dynamics, statistical mechanics, and hybrid quantum mechanics/molecular mechanics simulations to describe mechanistically the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp). Our study analyzes the binding mode of both natural triphosphate substrates as well as remdesivir triphosphate (the active form of drug), which is bound preferentially over ATP by RdRp while being poorly recognized by human RNA polymerase II (RNA Pol II). A comparison of incorporation rates between natural and antiviral nucleotides shows that remdesivir is incorporated more slowly into the nascent RNA compared with ATP, leading to an RNA duplex that is structurally very similar to an unmodified one, arguing against the hypothesis that remdesivir is a competitive inhibitor of ATP. We characterize the entire mechanism of reaction, finding that viral RdRp is highly processive and displays a higher catalytic rate of incorporation than human RNA Pol II. Overall, our study provides the first detailed explanation of the replication mechanism of RdRp.

Binding properties of [Ru(phen)2(11-R-dppz)]2+ (R = F or CN) with poly(A)•poly(U) duplex RNA

Two Ru(II) complexes, [Ru(phen)2(11-F-dppz)]2+ (Ru1, phen = 1,10-phenanthroline, 11-F-dppz = 11-fluorodipyrido[3,2-a:2′,3′-c]phenazine) and [Ru(phen)2(11-CN-dppz)]2+ (Ru2, 11-CN-dppz = 11-cyanodipyrido[3,2-a:2′,3′-c]phenazine), have been synthesized and characterized in this work. The binding properties of Ru1 and Ru2 with poly(A)•poly(U) RNA duplex have been investigated by spectroscopic methods and viscosity measurements. UV–vis absorption spectra and viscosity experiments demonstrate that the binding modes of Ru1 and Ru2 with poly(A)•poly(U) RNA duplex are intercalation, while the binding affinity for Ru2 is greater than that for Ru1. In addition, thermal denaturation studies reveal that both complexes significantly improve the stability of poly(A)•poly(U) duplex RNA. However, fluorescence titrations indicate that Ru1, unlike Ru2, can act as a molecular “light switch” for the poly(A)•poly(U) duplex RNA. The obtained results of this work indicate that the electron-withdrawing effect of substituents on the main ligands can significantly affect the binding of Ru(II) polypyridyl complexes with poly(A)•poly(U).

The role of siRNA in inhibition the biofilm formation as a first line of antibiotic resistance by regulation the MsrA drug efflux pump in Staphylococcus saprophyticus

The bacterium Staphylococcus saprophyticus (S. saprophyticus) is a common cause of urinary tract infections (UTI) in the community. To determine the effect of siRNA on the mRNA of the msrA gene in S. saprophyticus, 21-23 bp small interfering RNA (siRNA) duplexes were constructed against the mRNA of the msrA gene. The effect of siRNA on msrA mRNA expression was determined using reverse transcription PCR (RT-PCR). To assess changes in biofilm formation (BF) in response to siRNA activity, the usual tube technique was adopted. In vitro, msrA-siRNAs inhibited S. saprophyticus mRNA expression and activity. The efficacy of siRNA was determined by comparing the BF in S. saprophyticus before and after msrA-siRNA was introduced into the bacteria. In this investigation, two msrA-siRNA sequences, siRNA1 and siRNA2, were employed. qRT-PCR revealed that two msrA-siRNA sequences significantly suppressed the expression of msrA-mRNA, P = (0.010 and 0.002 ) respectively at (P< 0.05) comparison with control. Regarding the BF results after treatment with two siRNA sequences, they were 5/6 (83.5%) and 4/6 (67%) negative formation and 1/6 (16.5%) and 2/6 (33%) positive formation, respectively compared with control.

The Role of DEAD-Box ATPases in Gene Expression and the Regulation of RNA-Protein Condensates.

DEAD-box ATPases constitute a very large protein family present in all cells, often in great abundance. From bacteria to humans, they play critical roles in many aspects of RNA metabolism, and due to their widespread importance in RNA biology, they have been characterized in great detail at both the structural and biochemical levels. DEAD-box proteins function as RNA-dependent ATPases that can unwind short duplexes of RNA, remodel ribonucleoprotein (RNP) complexes, or act as clamps to promote RNP assembly. Yet, it often remains enigmatic how individual DEAD-box proteins mechanistically contribute to specific RNA-processing steps. Here, we review the role of DEAD-box ATPases in the regulation of gene expression and propose that one common function of these enzymes is in the regulation of liquid-liquid phase separation of RNP condensates.

PncCCND1_B Engages an Inhibitory Protein Network to Downregulate CCND1 Expression upon DNA Damage.

Simple SummaryEwing sarcoma is a pediatric tumor characterized by chromosomal translocations, giving rise to the oncogene EWS-FLI1, which triggers the transcription of genes involved in neoplastic transformation including CCND1. In this work, we found that exposure to etoposide, a topoisomerase II inhibitor usually administered in combination with other drugs in the standard regimen for Ewing sarcoma treatment, induced cell death and reduced CCND1 levels. Etoposide acts, at least in part, by enhancing the expression of the pncCCND1_B, a promoter-associated noncoding RNA transcribed from the promoter region of the CCND1 gene. PncCCND1_B regulates in cis CCND1 expression by forming a molecular complex with the RNA binding protein Sam68 and the DNA/RNA helicase DHX9. Upon exposure to etoposide, the increase in pncCCND1_B coupled with the decrease in DHX9 expression promote epigenetic changes and formation of DNA:RNA hybrids at the promoter region of CCND1 gene, which downregulate its expression.Promoter-associated noncoding RNAs (pancRNAs) represent a class of noncoding transcripts driven from the promoter region of protein-coding or non-coding genes that operate as cis-acting elements to regulate the expression of the host gene. PancRNAs act by altering the chromatin structure and recruiting transcription regulators. PncCCND1_B is driven by the promoter region of CCND1 and regulates CCND1 expression in Ewing sarcoma through recruitment of a multi-molecular complex composed of the RNA binding protein Sam68 and the DNA/RNA helicase DHX9. In this study, we investigated the regulation of CCND1 expression in Ewing sarcoma cells upon exposure to chemotherapeutic drugs. Pan-inhibitor screening indicated that etoposide, a drug used for Ewing sarcoma treatment, promotes transcription of pncCCND1_B and repression of CCND1 expression. RNA immunoprecipitation experiments showed increased binding of Sam68 to the pncCCND1_B after treatment, despite the significant reduction in DHX9 protein. This effect was associated with the formation of DNA:RNA duplexes at the CCND1 promoter. Furthermore, Sam68 interacted with HDAC1 in etoposide treated cells, thus contributing to chromatin remodeling and epigenetic changes. Interestingly, inhibition of the ATM signaling pathway by KU 55,933 treatment was sufficient to inhibit etoposide-induced Sam68-HDAC1 interaction without rescuing DHX9 expression. In these conditions, the DNA:RNA hybrids persist, thus contributing to the local chromatin inactivation at the CCND1 promoter region. Altogether, our results show an active role of Sam68 in DNA damage signaling and chromatin remodeling on the CCND1 gene by fine-tuning transitions of epigenetic complexes on the CCND1 promoter.

Multivalent Cations Reverse the Twist-Stretch Coupling of RNA.

When stretched, both DNA and RNA duplexes change their twist angles through twist-stretch coupling. The coupling is negative for DNA but positive for RNA, which is not yet completely understood. Here, our magnetic tweezers experiments show that the coupling of RNA reverses from positive to negative by multivalent cations. Combining with the previously reported tension-induced negative-to-positive coupling reversal of DNA, we propose a unified mechanism of the couplings of both RNA and DNA based on molecular dynamics simulations. Two deformation pathways are competing when stretched: shrinking the radius causes positive couplings but widening the major groove causes negative couplings. For RNA whose major groove is clamped by multivalent cations and canonical DNA, their radii shrink when stretched, thus exhibiting positive couplings. For elongated DNA whose radius already shrinks to the minimum and canonical RNA, their major grooves are widened when stretched, thus exhibiting negative couplings.

Synthesis of Crosslinked 2'-OMe RNA Duplexes Using 2-Amino-6-Vinylpurine and Their Application for Effective Inhibition of miRNA Function.

Crosslinking reactions to nucleic acids are an effective way to prepare stable complexes formed by covalent bonding. We demonstrated that fully 2'-O-methylated (2'-OMe) RNAs having a 2-amino-6-vinylpurine (AVP) exhibited an efficient crosslinking to uracil in the target RNA. Recently, we reported the preparation of crosslinked 2'-OMe RNA duplexes using AVP and the anti-miRNA oligonucleotides (AMOs) containing crosslinked duplexes at the terminal positions. These AMOs exhibited efficient microRNA (miRNA) inhibition at very low concentrations. In this article, we describe the chemical synthesis of 2'-OMe oligonucleotides containing AVP and preparation of the AMOs bearing crosslinked 2'-OMe RNA duplexes using AVP. In addition, we describe in detail the miRNA inhibition assay using these AMOs. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of phosphoramidite of 2-amino-6-vinylguanosine derivative Basic Protocol 2: Synthesis of AVP-2'-OMe RNA Basic Protocol 3: Evaluation of the crosslink reactivity of CFO containing AVP to the 2'-OMe RNA and preparation of AMOs containing crosslinked duplex Basic Protocol 4: miRNA inhibition assays.

Recognition of ATT Triplex and DNA:RNA Hybrid Structures by Benzothiazole Ligands.

Interactions of an array of nucleic acid structures with a small series of benzothiazole ligands (bis-benzothiazolyl-pyridines—group 1, 2-thienyl/2-benzothienyl-substituted 6-(2-imidazolinyl)benzothiazoles—group 2, and three 2-aryl/heteroaryl-substituted 6-(2-imidazolinyl)benzothiazoles—group 3) were screened by competition dialysis. Due to the involvement of DNA:RNA hybrids and triplex helices in many essential functions in cells, this study’s main aim is to detect benzothiazole-based moieties with selective binding or spectroscopic response to these nucleic structures compared to regular (non-hybrid) DNA and RNA duplexes and single-stranded forms. Complexes of nucleic acids and benzothiazoles, selected by this method, were characterized by UV/Vis, fluorescence and circular dichroism (CD) spectroscopy, isothermal titration calorimetry, and molecular modeling. Two compounds (1 and 6) from groups 1 and 2 demonstrated the highest affinities against 13 nucleic acid structures, while another compound (5) from group 2, despite lower affinities, yielded higher selectivity among studied compounds. Compound 1 significantly inhibited RNase H. Compound 6 could differentiate between B- (binding of 6 dimers inside minor groove) and A-type (intercalation) helices by an induced CD signal, while both 5 and 6 selectively stabilized ATT triplex in regard to AT duplex. Compound 3 induced strong condensation-like changes in CD spectra of AT-rich DNA sequences.

Insights into Binding of Single-Stranded Viral RNA Template to the Replication-Transcription Complex of SARS-CoV-2 for the Priming Reaction from Molecular Dynamics Simulations.

A minimal replication–transcription complex (RTC) of SARS-CoV-2 for synthesis of viral RNAs includes the nsp12 RNA-dependent RNA polymerase and two nsp8 RNA primase subunits for de novo primer synthesis, one nsp8 in complex with its accessory nsp7 subunit and the other without it. The RTC is responsible for faithfully copying the entire (+) sense viral genome from its first 5′-end to the last 3′-end nucleotides through a replication-intermediate (RI) template. The single-stranded (ss) RNA template for the RI is its 33-nucleotide 3′-poly(A) tail adjacent to a well-characterized secondary structure. The ssRNA template for viral transcription is a 5′-UUUAU-3′ next to stem-loop (SL) 1′. We analyze the electrostatic potential distribution of the nsp8 subunit within the RTC around the template strand of the primer/template (P/T) RNA duplex in recently published cryo-EM structures to address the priming reaction using the viral poly(A) template. We carried out molecular dynamics (MD) simulations with a P/T RNA duplex, the viral poly(A) template, or a generic ssRNA template. We find evidence that the viral poly(A) template binds similarly to the template strand of the P/T RNA duplex within the RTC, mainly through electrostatic interactions, providing new insights into the priming reaction by the nsp8 subunit within the RTC, which differs significantly from the existing proposal of the nsp7/nsp8 oligomer formed outside the RTC. High-order oligomerization of nsp8 and nsp7 for SARS-CoV observed outside the RTC of SARS-CoV-2 is not found in the RTC and not likely to be relevant to the priming reaction.

Photo-Cross-Linking between Br U and Pyrene Residues in an RNA/DNA Hybrid.

In this study, we investigated the photoreaction of Br U in a pyrene-labeled DNA duplex, RNA duplex, and DNA/RNA hybrids. We found that the photoreactivity of Br U changed dramatically from hydrogen abstraction to cross-linking by changing the conformation of the duplex from the B-form to the A-form. Among three A-form structures, the largest amount of cross-linked products was observed when Br U was incorporated into the RNA strand and the pyrene was conjugated to the 5' end of the DNA. These results indicate that the contact manner of pyrene was different between A- and B-form duplexes. This is a rare example of the use of the reactivity of bromouracil to analyze the contact between a small molecule with a weak binding affinity and a nucleic acid.

Rolling circle RNA synthesis catalyzed by RNA.

RNA-catalyzed RNA replication is widely considered a key step in the emergence of life's first genetic system. However, RNA replication can be impeded by the extraordinary stability of duplex RNA products, which must be dissociated for re-initiation of the next replication cycle. Here, we have explored rolling circle synthesis (RCS) as a potential solution to this strand separation problem. We observe sustained RCS by a triplet polymerase ribozyme beyond full-length circle synthesis with strand displacement yielding concatemeric RNA products. Furthermore, we show RCS of a circular Hammerhead ribozyme capable of self-cleavage and re-circularization. Thus, all steps of a viroid-like RNA replication pathway can be catalyzed by RNA alone. Finally, we explore potential RCS mechanisms by molecular dynamics simulations, which indicate a progressive build-up of conformational strain upon RCS with destabilization of nascent strand 5'- and 3'-ends. Our results have implications for the emergence of RNA replication and for understanding the potential of RNA to support complex genetic processes.

PCP Protein Inversin Regulates Testis Function Through Changes in Cytoskeletal Organization of Actin and Microtubules.

Inversin is an integrated component of the Frizzled (Fzd)/Dishevelled (Dvl)/Diversin planar cell polarity (PCP) complex that is known to work in concert with the Van Gogh-like protein (eg, Vangl2)/Prickle PCP complex to support tissue and organ development including the brain, kidney, pancreas, and others. These PCP protein complexes are also recently shown to confer developing haploid spermatid PCP to support spermatogenesis in adult rat testes. However, with the exception of Dvl3 and Vangl2, other PCP proteins have not been investigated in the testis. Herein, we used the technique of RNA interference (RNAi) to examine the role of inversin (Invs) in Sertoli cell (SC) and testis function by corresponding studies in vitro and in vivo. When inversin was silenced by RNAi using specific small interfering RNA duplexes by transfecting primary cultures of SCs in vitro or testes in vivo, it was shown that inversin knockdown (KD) perturbed the SC tight junction-barrier function in vitro and in vivo using corresponding physiological and integrity assays. More important, inversin exerted its regulatory effects through changes in the organization of the actin and microtubule cytoskeletons, including reducing the ability of their polymerization. These changes, in turn, induced defects in spermatogenesis by loss of spermatid polarity, disruptive distribution of blood-testis barrier-associated proteins at the SC-cell interface, appearance of multinucleated round spermatids, and defects in the release of sperm at spermiation.

The cap-proximal RNA secondary structure inhibits preinitiation complex formation on HAC1 mRNA

Translation of HAC1 mRNA in the budding yeast Saccharomyces cerevisiae is derepressed when RNase Ire1 removes its intron via nonconventional cytosolic splicing in response to accumulation of unfolded proteins inside the endoplasmic reticulum. The spliced HAC1 mRNA is translated into a transcription factor that changes the cellular gene expression patterns to increase the protein folding capacity of cells. Previously, we showed that a segment of the intronic sequence interacts with the 5′-UTR of the unspliced mRNA, resulting in repression of HAC1 translation at the initiation stage. However, the exact mechanism of translational derepression is not clear. Here, we show that at least 11-base-pairing interactions between the 5′-UTR and intron (UI) are sufficient to repress HAC1 translation. We also show that overexpression of the helicase eukaryotic initiation factor 4A derepressed translation of an unspliced HAC1 mRNA containing only 11-bp interactions between the 5′-UTR and intronic sequences. In addition, our genetic screen identifies that single mutations in the UI interaction site could derepress translation of the unspliced HAC1 mRNA. Furthermore, we show that the addition of 24 RNA bases between the mRNA 5′-cap and the UI interaction site derepressed translation of the unspliced HAC1 mRNA. Together, our data provide a mechanistic explanation for why the cap-proximal UI–RNA duplex inhibits the recruitment of translating ribosomes to HAC1 mRNA, thus keeping mRNA translationally repressed.