7SL RNA Research Articles

Sequential structure probing of cotranscriptional RNA folding intermediates.

Cotranscriptional RNA folding pathways typically involve the sequential formation of folding intermediates. Existing methods for cotranscriptional RNA structure probing map the structure of nascent RNA in the context of a terminally arrested transcription elongation complex. Consequently, the rearrangement of RNA structures as nucleotides are added to the transcript can be inferred but is not assessed directly. To address this limitation, we have developed linked- m ultipoint T ranscription E longation C omplex RNA structure prob ing (TECprobe-LM), which assesses the cotranscriptional rearrangement of RNA structures by sequentially positioning E. coli RNAP at two or more points within a DNA template so that nascent RNA can be chemically probed. We validated TECprobe-LM by measuring known folding events that occur within the E. coli signal recognition particle RNA, Clostridium beijerinckii pfl ZTP riboswitch, and Bacillus cereus crcB fluoride riboswitch folding pathways. Our findings establish TECprobe-LM as a strategy for detecting cotranscriptional RNA folding events directly using chemical probing.

Regulation of transcription by RNA polymerase III promotors in norm and pathology

RNA polymerase III synthesizes a wide range of non-coding RNAs shorter than 400 nucleotides in length. These RNAs are involved in protein synthesis (tRNA, 5S rRNA, and 7SL RNA), maturation and splicing of different types of RNA (RPR, MRP RNA, and U6 snRNA), regulation of transcription (7SK RNA), replication (Y RNA), and intracellular transport (vault RNA). BC200 and BC1 RNA genes are transcribed by RNA polymerase III in neurons only where these RNAs regulate protein synthesis. Mutations in the regulatory elements of the genes transcribed by RNA polymerase III as well as in transcription factors of this RNA polymerase are associated with the development of a number of diseases, primarily oncological and neurological. In this regard, the mechanisms of regulation of the expression of the genes containing various RNA polymerase III promoters were actively studied. This review describes the structural and functional classification of polymerase III promoters, as well as the factors involved in the regulation of promoters of different types. A number of examples demonstrate the role of the described factors in the pathogenesis of human diseases.

Signal recognition particle RNA is critical for genetic competence and virulence of Streptococcus pneumoniae.

Streptococcus pneumoniae (pneumococcus) causes multiple important infectious diseases in humans. The signal recognition particle (SRP) complex, which comprised 4.5S small cytoplasmic RNA (ScRNA), and the Ffh and FtsY proteins, mediates membrane targeting and translocation of secreted proteins in all organisms. However, the role of SRP and ScRNA has not been characterized during the induction of the competence system for genetic transformation and virulence in pneumococcus. By using a combination of genetic, biochemical, proteomic, and imaging approaches, we demonstrated that the SRP complex plays a significant role in membrane targeting of competence system-regulated effectors important for genetic transformation, virulence during bacteremia and pneumonia infections, and nutritional acquisition.

Deciphering the Binding of 5' Stem Loop RNA to the La Domain of Human LARP6.

La-related protein 6 regulates the highly organized biosynthesis of type I procollagen polypeptides and affects proper assembly of procollagen peptides into heterotrimers of type I procollagen. LARP6-mediated regulation of collagen biosynthesis is mediated through interaction with the 5' stem loop motif found in type I and III collagen mRNA. Recent studies highlight the involvement of HsLARP6 in fibroproliferative diseases and its potential as a target for therapeutic intervention. The intrinsic instability of the La domain of HsLARP6 hampers studies probing the molecular basis of biologically- and disease-relevant structure-function relationship, particularly when high concentrations are required. This work provides detailed procedures to produce milligram amounts of RNase-free and functional La domain of HsLARP6. Furthermore, we investigated the effect of the construct length as well as RNA binding on protein stability. N- and C-terminal extensions greatly impact stability based on interactions with the core domain and modulation of the pI. When in complex with its cognate 5'SL RNA, the La domain shows unprecedented stability compared to the aggregation-prone unbound state. The protein-RNA complex remains stable for at least 50x longer than the unbound state, under identical conditions, likely due to a global change in conformational plasticity upon RNA binding. These results provide a foundation for further studies of the molecular recognition of 5'SL by HsLARP6 as well as a platform for refining potential antifibrotic therapeutics.

Variable patterns of retrotransposition in different HeLa strains provide mechanistic insights into SINE RNA mobilization processes.

Alu elements are non-autonomous Short INterspersed Elements (SINEs) derived from the 7SL RNA gene that are present at over one million copies in human genomic DNA. Alu mobilizes by a mechanism known as retrotransposition, which requires the Long INterspersed Element-1 (LINE-1) ORF2-encoded protein (ORF2p). Here, we demonstrate that HeLa strains differ in their capacity to support Alu retrotransposition. Human Alu elements retrotranspose efficiently in HeLa-HA and HeLa-CCL2 (Alu-permissive) strains, but not in HeLa-JVM or HeLa-H1 (Alu-nonpermissive) strains. A similar pattern of retrotransposition was observed for other 7SL RNA-derived SINEs and tRNA-derived SINEs. In contrast, mammalian LINE-1s, a zebrafish LINE, a human SINE-VNTR-Alu (SVA) element, and an L1 ORF1-containing mRNA can retrotranspose in all four HeLa strains. Using an in vitro reverse transcriptase-based assay, we show that Alu RNAs associate with ORF2p and are converted into cDNAs in both Alu-permissive and Alu-nonpermissive HeLa strains, suggesting that 7SL- and tRNA-derived SINEs use strategies to 'hijack' L1 ORF2p that are distinct from those used by SVA elements and ORF1-containing mRNAs. These data further suggest ORF2p associates with the Alu RNA poly(A) tract in both Alu-permissive and Alu-nonpermissive HeLa strains, but that Alu retrotransposition is blocked after this critical step in Alu-nonpermissive HeLa strains.

Effects of PNA Sequence and Target Site Selection on Function of a 4.5S Non-Coding RNA.

Peptide nucleic acid (PNA) based antisense strategy is a promising therapeutic approach to specifically inhibit target gene expression. However, unlike protein coding genes, identification of an ideal PNA binding site for non-coding RNA is not straightforward. Here, we compare the inhibitory activities of PNA molecules that bind a non-coding 4.5S RNA called SRP RNA, a key component of the bacterial signal recognition particle (SRP). A 9-mer PNA (PNA9) complementary to the tetraloop region of the RNA was more potent in inhibiting its interaction with the SRP protein, compared to an 8-mer PNA (PNA8) targeting a stem-loop. PNA9, which contained a homo-pyrimidine sequence could form a triplex with the complementary stretch of RNA in vitro as confirmed using a fluorescent derivative of PNA9 (F-PNA13). The RNA-PNA complex formation resulted in inhibition of SRP function with PNA9 and F-PNA13, but not PNA8 highlighting the importance of target site selection. Surprisingly, F-PNA13 which was more potent in inhibiting SRP function in vitro, showed weaker antibacterial activity compared to PNA9 likely due to poor cell penetration of the longer PNA. Our results underscore the importance of suitable target site selection and optimum PNA length to develop better antisense molecules against non-coding RNA.

Regulation of Transcription by RNA Polymerase III Promotors in the Norm and Pathology

RNA polymerase III synthesizes a wide range of noncoding RNAs shorter than 400 nucleotides in length. These RNAs are involved in protein synthesis (tRNA, 5S rRNA, and 7SL RNA), maturation, and splicing of different types of RNA (RPR, MRP RNA, and U6 snRNA), regulation of transcription (7SK RNA), replication (Y RNA), and intracellular transport (vault RNA). BC200 and BC1 RNA genes are transcribed by RNA polymerase III in neurons only where these RNAs regulate protein synthesis. Mutations in the regulatory elements of the genes transcribed by RNA polymerase III as well as in transcription factors of this RNA polymerase are associated with the development of a number of diseases, primarily oncological and neurological. In this regard, the mechanisms of regulation of the expression of the genes containing various RNA polymerase III promoters were actively studied. This review describes the structural and functional classification of polymerase III promoters, as well as the factors involved in the regulation of promoters of different types. A number of examples demonstrate the role of the described factors in the pathogenesis of human diseases.

The Accuracy of Diagnosis and Genotyping of Leishmania Species Based on Spliced Leader Mini-Exon Gene by Nuclear Magnetic Resonance and Sequencing Assays.

We aimed to evaluate the accuracy of genotyping of Leishmania species by the spliced leader mini-exon gene. Suspected leishmaniasis patients, referred to Masieh Daneshvary Hospital, Tehran, Iran were included from May 2017 to September 2021. The Leishmania species were genotyped by PCRRFLP based on the SL mini-exon gene and the ITS1 region of SSU-rRNA gene and compared with the sequencing results. The expressed metabolites of metacyclic promastigotes were evaluated by Proton nuclear magnetic resonance (1H-NMR). Out of 66 suspected cases, 36 (54.4%) were positive for Leishmania species based on the PCR assays. In 21 (31.8%) cases, promastigotes grew on culture tubes. Based on the RFLP of SL RNA profile, 13 (19.7%) L. tropica, 9 (13.6%) L. major, 3 (4.5%) L. infantum, and 8 (12.1%) C. fasciculata isolates, isolated from culture media, were identified; however, 3 (4.5%) cases were unidentifiable due to the low number of parasites. Seventeen metabolites were expressed by the metacyclic forms of L. major, L. tropica and C. fasciculata isolates. The top differential metabolites expressed more in C. fasciculata were FAD, p-Methoxybenzyl alcohol and S-b-G-5, 5-G-b-S (A = CH2) (P<0.005) whereas Veratryl glycerols and D-(+)-Mannose were significantly increased in L. major and Betulin, LTyrosine in L. tropica (P<0.01). The invaluable techniques such as sequencing and 1H-NMR confirmed the results of genotyping of Leishmania species based on the SL mini-exon gene. SL mini exon gene can be used as a diagnostic tool to differentiate various Leishmania genotypes and detect contamination of culture media with C. fasciculata.

Detection of a 7SL RNA-derived small non-coding RNA using Molecular Beacons invitro and in cells.

Small non-coding RNAs (sncRNA) are involved in many steps of the gene expression cascade and regulate processing and expression of mRNAs by the formation ofribonucleoprotein complexes (RNP) such as the RNA-induced silencing complex (RISC). By analyzing small RNA Seq data sets, we identified a sncRNA annotated as piR-hsa-1254, which is likely derived from the 3'-end of 7SL RNA2 (RN7SL2), herein referred to as snc7SL RNA. The 7SL RNA is an abundant long non-coding RNA polymerase III transcript and serves as structural component of the cytoplasmic signal recognition particle (SRP). To evaluate a potential functional role of snc7SL RNA, we aimed to define its cellular localization by live cell imaging. Therefore, a Molecular Beacon (MB)-based method was established to compare the subcellular localization of snc7SL RNA with its precursor 7SL RNA. We designed and characterized several MBs invitro and tested those by live cell fluorescence microscopy. Using a multiplex approach, we show that 7SL RNA localizes mainly to the endoplasmic reticulum (ER), as expected for the SRP, whereas snc7SL RNA predominately localizes to the nucleus. This finding suggests a fundamentally different function of 7SL RNA and its derivate snc7SL RNA.

Nuclear SRP9/SRP14 heterodimer transcriptionally regulates 7SL and BC200 RNA expression.

The SRP9/SRP14 heterodimer is a central component of signal recognition particle (SRP) RNA (7SL) processing and Alu retrotransposition. In this study, we sought to establish the role of nuclear SRP9/SRP14 in the transcriptional regulation of 7SL and BC200 RNA. 7SL and BC200 RNA steady-state levels, rate of decay, and transcriptional activity were evaluated under SRP9/SRP14 knockdown conditions. Immunofluorescent imaging, and subcellular fractionation of MCF-7 cells, revealed a distinct nuclear localization for SRP9/SRP14. The relationship between this localization and transcriptional activity at 7SL and BC200 genes was also examined. These findings demonstrate a novel nuclear function of SRP9/SRP14 establishing that this heterodimer transcriptionally regulates 7SL and BC200 RNA expression. We describe a model in which SRP9/SRP14 cotranscriptionally regulate 7SL and BC200 RNA expression. Our model is also a plausible pathway for regulating Alu RNA transcription and is consistent with the hypothesized roles of SRP9/SRP14 transporting 7SL RNA into the nucleolus for posttranscriptional processing, and trafficking of Alu RNA for retrotransposition.

Nucleotide Context Can Modulate Promoter Strength in Genes Transcribed by RNA Polymerase III.

The small nuclear RNAs 4.5SH and 4.5SI were characterized only in mouse-like rodents; their genes originate from 7SL RNA and tRNA, respectively. Similar to many genes transcribed by RNA polymerase III (pol III), the genes of 4.5SH and 4.5SI RNAs include boxes A and B, forming an intergenic pol III-directed promoter. In addition, their 5'-flanking sequences have TATA-like boxes at position -31/-24, also required for efficient transcription. The patterns of the three boxes notably differ in the 4.5SH and 4.5SI RNA genes. The A, B, and TATA-like boxes were replaced in the 4.5SH RNA gene with the corresponding boxes in the 4.5SI RNA gene to evaluate their effect on the transcription of transfected constructs in HeLa cells. Simultaneous replacement of all three boxes decreased the transcription level by 40%, which indicates decreased promoter activity in a foreign gene. We developed a new approach to compare the promoter strength based on the competition of two co-transfected gene constructs when the proportion between the constructs modulates their relative activity. This method demonstrated that the promoter activity of 4.5SI is 12 times that of 4.5SH. Unexpectedly, the replacement of all three boxes of the weak 4.5SH promoter with those of the strong 4.5SI gene significantly reduced, rather than enhanced, the promoter activity. Thus, the strength of a pol III-directed promoter can depend on the nucleotide environment of the gene.

Inhibition of RNA Polymerase III Augments the Anti-Cancer Properties of TNFα

Tumour necrosis factor alpha (TNFα) is a multifunctional cytokine that plays a pivotal role in apoptosis, cell survival, as well as in inflammation and immunity. Although named for its antitumor properties, TNFα also has tumour-promoting properties. TNFα is often present in large quantities in tumours, and cancer cells frequently acquire resistance to this cytokine. Consequently, TNFα may increase the proliferation and metastatic potential of cancer cells. Furthermore, the TNFα-driven increase in metastasis is a result of the ability of this cytokine to induce the epithelial-to-mesenchymal transition (EMT). Overcoming the resistance of cancer cells to TNFα may have a potential therapeutic benefit. NF-κB is a crucial transcription factor mediating inflammatory signals and has a wide-ranging role in tumour progression. NF-κB is strongly activated in response to TNFα and contributes to cell survival and proliferation. The pro-inflammatory and pro-survival function of NF-κB can be disrupted by blocking macromolecule synthesis (transcription, translation). Consistently, inhibition of transcription or translation strongly sensitises cells to TNFα-induced cell death. RNA polymerase III (Pol III) synthesises several essential components of the protein biosynthetic machinery, such as tRNA, 5S rRNA, and 7SL RNA. No studies, however, directly explored the possibility that specific inhibition of Pol III activity sensitises cancer cells to TNFα. Here we show that in colorectal cancer cells, Pol III inhibition augments the cytotoxic and cytostatic effects of TNFα. Pol III inhibition enhances TNFα-induced apoptosis and also blocks TNFα-induced EMT. Concomitantly, we observe alterations in the levels of proteins related to proliferation, migration, and EMT. Finally, our data show that Pol III inhibition is associated with lower NF-κB activation upon TNFα treatment, thus potentially suggesting the mechanism of Pol III inhibition-driven sensitisation of cancer cells to this cytokine.

Non-coding RNAs and functional RNA elements in Thermus thermophilus.

To complete the ThermusQ database, small non-coding RNAs (ncRNAs) and functional RNA elements found in Thermus thermophilus were summarized with annotations. The well-known three ncRNAs, M1 RNA, tmRNA and SRP RNA, were annotated as ttj8_nc001 to ttj8_nc003, and 10 novel RNAs were annotated as ttj8_nc004 to ttj8_nc013. Antisense RNAs for some ORFs were annotated as ttj8_EST00001 to ttj8_EST00006. In addition, a set of conserved sequences found in T. thermophilus HB27 were also described.

A FAM46C-Dependent Molecular Circuit Modulates Antibody Synthesis and Proliferation in Plasma Cell Dyscrasias

Plasma cells (PCs) are the terminal effectors of B lymphocyte differentiation in charge of antibody production. Such intensive task requires rapid endoplasmic reticulum (ER) expansion and the concerted induction of the machinery affording ER import, folding, and trafficking along the secretory route. Moreover, the by-products of massive immunoglobulin (Ig) production cause a peculiar dependence of PCs on protein degradative pathways with a crucial clinical consequence: theextraordinary success of proteasome inhibitors against multiple myeloma (MM) and systemic light chain amyloidosis (AL). Notably, an essential player in the proteomic reshaping occurring during PC differentiation is the non-canonical poly(A)polymerase FAM46C/TENT5C. Upon B cell activation, FAM46C is induced to polyadenylate and stabilize mRNAs encoding Igs and ER-targeted proteins, thus promoting humoral immune responses (Mroczek et al., 2017; Bilska et al., 2020). Interestingly, FAM46C locus on chromosome 1p12 is mutated or deleted in up to 20% MM patients (Chapman et al., 2011; Boyd et al., 2011, Barbieri et al., 2016), suggesting that MM cells are subject to a selective pressure to restrict FAM46C expression and activity, to ultimately curb Ig synthesis and secretion in favor of cell survival and tumor growth. Accordingly, exogenous re-expression of FAM46C in mutated MM cells raised the secretory capacity beyond sustainability, resulting in intracellular ATP reduction, ROS accumulation and increased apoptotic susceptibility (Fucci et al., 2020). Our research aims to discover the precise molecular circuits, centered on FAM46C, regulating PC secretory capacity in efficient coordination with degradative pathways, to discover unprecedented vulnerabilities of PCs and new therapeutic opportunities against PC dyscrasias. To clarify the molecular mechanisms underlying the effects of FAM46C on Ig secretion we combined cutting-edge protein biochemistry, imaging and unbiased proteomic assays and adopted in vitro and in vivo models of MM and AL. In these models, we disclosed that FAM46C concertedly promotes the expression of ER and Golgi proteins, potently expanding the secretory apparatus and light chain (LC) secretion. To assess the impact of FAM46C on PC proliferation and disease progression in vivo, we silenced or overexpressed FAM46C in AMLC-2 cells that bear a hemizygous loss in 1p12 region (Arendt et al., 2008). Following intravenous injection into Rag2-/- IL2rg-/- mice, we found remarkably reduced tumor growth and longer survival in recipients of FAM46C-overexpressing cells, despite higher initial circulating LC levels, as compared to other groups. Moreover, we observed intermediate disease progression in mice receiving mock-transduced ALMC-2 cells, attesting to dose-dependent impact of FAM46C. The in vivo association of higher secretory activity with drastically decreased PC proliferation reveals that FAM46C uncouples monoclonal component levels from disease burden. Finally, among FAM46C-modulated proteins, we found SRP components and RNA methyltransferases involved in ribosome assembly and tRNA processing. The data unveil a new role played by FAM46C in PCs, in addition to its mRNA stabilizing activity, indicating the existence of a previously unrecognized integrated network coordinating ribosome biogenesis, protein translation, and ER expansion, potentially crucial to harmonize intensive protein secretion with cellular homeostasis. Altogether, our studies disclose an unexpected molecular circuit orchestrating Ig synthesis and secretion, and regulating PC proliferation and survival that may be exploited to design new therapeutic strategies aimed to reduce pathogenic LC production, to eliminate MM and AL cells, and to improve the efficacy of current therapeutic regimens.

5-methylcytosine (m5C) RNA modification controls the innate immune response to virus infection by regulating type I interferons

5-methylcytosine (m5C) is one of the most prevalent modifications of RNA, playing important roles in RNA metabolism, nuclear export, and translation. However, the potential role of RNA m5C methylation in innate immunity remains elusive. Here, we show that depletion of NSUN2, an m5C methyltransferase, significantly inhibits the replication and gene expression of a wide range of RNA and DNA viruses. Notably, we found that this antiviral effect is largely driven by an enhanced type I interferon (IFN) response. The antiviral signaling pathway is dependent on the cytosolic RNA sensor RIG-I but not MDA5. Transcriptome-wide mapping of m5C following NSUN2 depletion in human A549 cells revealed a marked reduction in the m5C methylation of several abundant noncoding RNAs (ncRNAs). However, m5C methylation of viral RNA was not noticeably altered by NSUN2 depletion. In NSUN2-depleted cells, the host RNA polymerase (Pol) III transcribed ncRNAs, in particular RPPH1 and 7SL RNAs, were substantially up-regulated, leading to an increase of unshielded 7SL RNA in cytoplasm, which served as a direct ligand for the RIG-I-mediated IFN response. In NSUN2-depleted cells, inhibition of Pol III transcription or silencing of RPPH1 and 7SL RNA dampened IFN signaling, partially rescuing viral replication and gene expression. Finally, depletion of NSUN2 in an ex vivo human lung model and a mouse model inhibits viral replication and reduces pathogenesis, which is accompanied by enhanced type I IFN responses. Collectively, our data demonstrate that RNA m5C methylation controls antiviral innate immunity through modulating the m5C methylome of ncRNAs and their expression.

Effect of pausing on the cotranscriptional folding kinetics of RNAs

The pausing event in RNA molecule folding that occurs during the cotranscription process plays a critical role in cellular RNA function. Based on Conformational Resampling through Kinetic Relaxation (CRKR), this paper investigates a method to reveal the specifics of pausing for RNA cotranscriptional folding of 117-nucleotide E. coli SRP RNA and 73-nucleotide HIV-1 TAR RNA. It can be inferred from the results that pausing events generate valid cotranscriptional conformational rearrangement to protect the function structures and influence the folding pathway, which is remarkably consistent with the experimental results. Additionally, different transcription speeds result in different levels of protection capability. The folding pathway or conformational rearrangement can also be affected by a change in transcription speed after pausing site. These findings illuminate that RNAs with functional structures undergo complex rearrangement folding in pausing events, and different RNAs prefer different speeds for specific folding pathways. Moreover, pausing in particular circumstances can be employed to regulate the population of final functional structures. In conclusion, this paper offers fresh perspectives on the pausing event in the cotranscriptional folding of RNAs.

Allosteric mechanism for SL RNA recognition by polypyrimidine tract binding protein RRM1: An atomistic MD simulation and network-based study

Polypyrimidine tract-binding protein (PTB), an RNA-binding protein, is involved in the regulation of diverse processes in mRNA metabolism. However, the allosteric modulation of its binding with RNA remains unclear. We explore the dynamic characteristics of PTB RNA recognition motif 1 (RRM1) in its RNA-free and wild-type/mutant RNA-bound states to understand the issues using molecular dynamics (MD) simulation, perturbation response scanning (PRS) and protein structure network (PSN) models. It is found that RNA binding strengthens RRM1 stability, while L151G mutation in α3 helix far away from the interface makes the complex unstable. The latter is caused by long-distance dynamic couplings, which makes intermolecular electrostatic and entropy energies unfavorable. The weakened couplings between interface β sheets and C-terminal parts upon mutation reveal RNA recognition is co-regulated by these regions. Interestingly, PRS analysis reveals the allostery caused by the perturbation on α3 helix has already been pre-encoded in the equilibrium dynamics of the protein structure. PSN analysis shows the details of the allosteric signal transmission, revealing the necessity of strong couplings between α3 helix and interface for maintaining the high binding affinity. This study sheds light on the mechanisms of PTB allostery and RNA recognition and can provide important information for drug design.

Spliceosomal SL1 RNA binding to U1-70K: the role of the extended RRM.

The RNA recognition motif (RRM) occurs widely in RNA-binding proteins, but does not always by itself support full binding. For example, it is known that binding of SL1 RNA to the protein U1-70K in the U1 spliceosomal particle is reduced when a region flanking the RRM is truncated. How the RRM flanking regions that together with the RRM make up an ‘extended RRM’ (eRRM) contribute to complex stability and structural organization is unknown. We study the U1-70K eRRM bound to SL1 RNA by thermal dissociation and laser temperature jump kinetics; long-time molecular dynamics simulations interpret the experiments with atomistic resolution. Truncation of the helix flanking the RRM on its N-terminal side, ‘N-helix,’ strongly reduces overall binding, which is further weakened under higher salt and temperature conditions. Truncating the disordered region flanking the RRM on the C-terminal side, ‘C-IDR’, affects the local binding site. Surprisingly, all-atom simulations show that protein truncation enhances base stacking interactions in the binding site and leaves the overall number of hydrogen bonds intact. Instead, the flanking regions of the eRRM act in a distributed fashion via collective interactions with the RNA when external stresses such as temperature or high salt mimicking osmotic imbalance are applied.

Identification and functional implications of pseudouridine RNA modification on small noncoding RNAs in the mammalian pathogen Trypanosoma brucei

Trypanosoma brucei, the parasite that causes sleeping sickness, cycles between an insect and a mammalian host. However, the effect of RNA modifications such as pseudouridinylation on its ability to survive in these two different host environments is unclear. Here, two genome-wide approaches were applied for mapping pseudouridinylation sites (Ψs) on small nucleolar RNA (snoRNA), 7SL RNA, vault RNA, and tRNAs from T. brucei. We show using HydraPsiSeq and RiboMeth-seq that the Ψ on C/D snoRNA guiding 2′-O-methylation increased the efficiency of the guided modification on its target, rRNA. We found differential levels of Ψs on these noncoding RNAs in the two life stages (insect host and mammalian host) of the parasite. Furthermore, tRNA isoform abundance and Ψ modifications were characterized in these two life stages demonstrating stage-specific regulation. We conclude that the differential Ψ modifications identified here may contribute to modulating the function of noncoding RNAs involved in rRNA processing, rRNA modification, protein synthesis, and protein translocation during cycling of the parasite between its two hosts.

The SNAPc complex mediates starvation-induced trans-splicing in Caenorhabditis elegans

Dietary restriction usually suppresses biosynthesis but activates catabolic pathways in animals. However, the short-term starvation enhances biosynthetic activities and promotes ribosomal biogenesis in adult Caenorhabditis elegans. The mechanism underlying the processes remains largely unknown. Here, we find that the short-term starvation enhances the SL1 trans-splicing of translation-related genes in adult C. elegans by transcriptome analysis. The small nuclear RNA-activating protein complex (SNAPc) promotes SL RNA production and mediates starvation-induced trans-splicing. TOFU-5, a core factor in the upstream sequence transcription complex (USTC) essential for piRNA production, is also involved in the starvation-induced trans-splicing processes. Knocking down components of the SNAPc complex and tofu-5 extends worm survival under starvation conditions. Taken together, our study highlights the importance of SL trans-splicing in the nutrition response and reveals a mechanism of the survival regulation by food deprivation via SNAPc and TOFU-5.