Sequence-encoded and composition-dependent protein-RNA interactions control multiphasic condensate morphologies.
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ABSTRACT:
SUBMITTER: Kaur T
PROVIDER: S-EPMC7870978 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Many eukaryotic organisms encode more than one RNA-dependent RNA polymerase (RdRP) that probably emerged as a result of gene duplication. Such RdRP paralogs often participate in distinct RNA silencing pathways and show characteristic repertoires of enzymatic activities in vitro However, to what extent members of individual paralogous groups can undergo functional changes during speciation remains an open question. We show that orthologs of QDE-1, an RdRP component of the quelling pathway in Neurospora crassa, have rapidly diverged in evolution at the amino acid sequence level. Analyses of purified QDE-1 polymerases from N. crassa (QDE-1(Ncr)) and related fungi, Thielavia terrestris (QDE-1(Tte)) and Myceliophthora thermophila (QDE-1(Mth)), show that all three enzymes can synthesize RNA, but the precise modes of their action differ considerably. Unlike their QDE-1(Ncr) counterpart favoring processive RNA synthesis, QDE-1(Tte) and QDE-1(Mth) produce predominantly short RNA copies via primer-independent initiation. Surprisingly, a 3.19 Å resolution crystal structure of QDE-1(Tte) reveals a quasisymmetric dimer similar to QDE-1(Ncr) Further electron microscopy analyses confirm that QDE-1(Tte) occurs as a dimer in solution and retains this status upon interaction with a template. We conclude that divergence of orthologous RdRPs can result in functional innovation while retaining overall protein fold and quaternary structure.
| S-EPMC4861493 | biostudies-literature
Project description:SHAPE-JuMP is a concise strategy for identifying close-in-space interactions in RNA molecules. Nucleotides in close three-dimensional proximity are crosslinked with a bi-reactive reagent that covalently links the 2'-hydroxyl groups of the ribose moieties. The identities of crosslinked nucleotides are determined using an engineered reverse transcriptase that jumps across crosslinked sites, resulting in a deletion in the cDNA that is detected using massively parallel sequencing. Here we introduce ShapeJumper, a bioinformatics pipeline to process SHAPE-JuMP sequencing data and to accurately identify through-space interactions, as observed in complex JuMP datasets. ShapeJumper identifies proximal interactions with near-nucleotide resolution using an alignment strategy that is optimized to tolerate the unique non-templated reverse-transcription profile of the engineered crosslink-traversing reverse-transcriptase. JuMP-inspired strategies are now poised to replace adapter-ligation for detecting RNA-RNA interactions in most crosslinking experiments.
| S-EPMC8670686 | biostudies-literature
Project description:BackgroundSequence determines the three-dimensional structure of RNAs, and thereby plays an important role in carrying out various biological functions. RNA duplexes containing Watson-Crick (WC) basepairs, interspersed with non-Watson-Crick basepairs, are the dominant structural unit and form the scaffold for the 3-dimensional structure of RNA. It is therefore crucial to understand the geometric variation in the dinucleotide steps that form the helices. We have carried out a detailed analysis of the dinucleotide steps formed by AU and GC Watson-Crick basepairs in RNA structures (both free and protein bound) and compared the results to that seen in DNA. Further, the effect of protein binding on these steps was examined by comparing steps in free RNA structures with protein bound RNA structures.ResultsCharacteristic sequence dependent geometries are observed for the RR, RY and YR type of dinucleotide steps in RNA. Their geometric parameters show correlated variations that are different from those observed in B-DNA helices. Subtle, but statistically significant differences are seen in roll, slide and average propeller-twist values, between the dinucleotide steps of free RNA and protein bound RNA structures. Many non-canonical cross-strand and intra-strand hydrogen bonds were identified that can stabilise the RNA dinucleotide steps, among which YR steps show presence of many new unreported interactions.ConclusionsOur work provides for the first time a detailed analysis of the conformational preferences exhibited by Watson-Crick basepair containing steps in RNA double helices. Overall, the WC dinucleotide steps show considerable conformational variability. Furthermore, we have identified hydrogen bond interactions in several of the dinucleotide steps that could play a role in determining the preferred geometry, in addition to the intra-basepair hydrogen bonds and stacking interactions. Protein binding affects the conformation of the steps that are in direct contact, as well as allosterically affect the steps that are not in direct physical contact.
| S-EPMC3930292 | biostudies-literature
Project description:BackgroundRNA-protein interactions (RPIs) play important roles in a wide variety of cellular processes, ranging from transcriptional and post-transcriptional regulation of gene expression to host defense against pathogens. High throughput experiments to identify RNA-protein interactions are beginning to provide valuable information about the complexity of RNA-protein interaction networks, but are expensive and time consuming. Hence, there is a need for reliable computational methods for predicting RNA-protein interactions.ResultsWe propose RPISeq, a family of classifiers for predicting RNA-protein interactions using only sequence information. Given the sequences of an RNA and a protein as input, RPIseq predicts whether or not the RNA-protein pair interact. The RNA sequence is encoded as a normalized vector of its ribonucleotide 4-mer composition, and the protein sequence is encoded as a normalized vector of its 3-mer composition, based on a 7-letter reduced alphabet representation. Two variants of RPISeq are presented: RPISeq-SVM, which uses a Support Vector Machine (SVM) classifier and RPISeq-RF, which uses a Random Forest classifier. On two non-redundant benchmark datasets extracted from the Protein-RNA Interface Database (PRIDB), RPISeq achieved an AUC (Area Under the Receiver Operating Characteristic (ROC) curve) of 0.96 and 0.92. On a third dataset containing only mRNA-protein interactions, the performance of RPISeq was competitive with that of a published method that requires information regarding many different features (e.g., mRNA half-life, GO annotations) of the putative RNA and protein partners. In addition, RPISeq classifiers trained using the PRIDB data correctly predicted the majority (57-99%) of non-coding RNA-protein interactions in NPInter-derived networks from E. coli, S. cerevisiae, D. melanogaster, M. musculus, and H. sapiens.ConclusionsOur experiments with RPISeq demonstrate that RNA-protein interactions can be reliably predicted using only sequence-derived information. RPISeq offers an inexpensive method for computational construction of RNA-protein interaction networks, and should provide useful insights into the function of non-coding RNAs. RPISeq is freely available as a web-based server at http://pridb.gdcb.iastate.edu/RPISeq/.
| S-EPMC3322362 | biostudies-literature
Project description:We exploit the unusual genome organization of the ciliate cell to analyze the control of specific gene amplification during a nuclear differentiation process. Ciliates contain two types of nuclei within one cell, the macronucleus and the micronucleus; and after sexual reproduction a new macronucleus is formed from a micronuclear derivative. During macronuclear differentiation, most extensive DNA reorganization, elimination, and fragmentation processes occur, resulting in a macronucleus containing short DNA molecules (nanochromosomes) representing individual genetic units and each being present in high copy number. It is believed that these processes are controlled by small nuclear RNAs but also by a template derived from the old macronucleus. We first describe the exact copy numbers of selected nanochromosomes in the macronucleus, and define the timing during nuclear differentiation at which copy number is determined. This led to the suggestion that DNA processing and copy number control may be closely related mechanisms. Degradation of an RNA template derived from the macronucleus leads to significant decrease in copy number, whereas injection of additional template molecules results in an increase in copy number and enhanced expression of the corresponding gene. These observations can be incorporated into a mechanistic model about an RNA-dependent epigenetic regulation of gene copy number during nuclear differentiation. This highlights that RNA, in addition to its well-known biological functions, can also be involved in the control of gene amplification.
| S-EPMC3009782 | biostudies-literature
Project description:BackgroundEukaryotes use RNA interference and RNA-based epigenetic regulation to control transposon activity. In the standard pathways of RNA-based transcriptional and post-transcriptional silencing the protein complex RNA-dependent RNA polymerase (RdRP) plays a crucial role. However, alternative pathways that bypass RdRP have recently been described. Hence two important questions are: is RdRP truly a necessary component for transposon control, and are the alternative RNA-based strategies also capable of controlling transposable elements?ResultsWe have studied the interplay between host RNAi pathways and transposons using mathematical models. We show that the canonical RdRP-based model controls transposons tightly, mainly via the feedback of cytoplasmic small RNA amplification. Next, we consider two variants lacking RdRP and instead employing antisense transcription of transposons. We show that transposon activity is also controlled by the alternative pathways, although cytoplasmic small RNA amplification is absent. Instead, control occurs in the nucleus, through a feedback in the epigenetic regulation.ConclusionsConcluding, our models show that the control of transposon activity can be achieved by alternative pathways that lack RdRP and act through different feedback mechanisms. Thus, although RdRP activity is ubiquitous in eukaryotes, it need not be a general requirement for transposon control.
| S-EPMC3155503 | biostudies-literature
Project description:RNA-binding proteins control many aspects of cellular biology through binding single-stranded RNA binding motifs (RBMs). However, RBMs can be buried within their local RNA structures, thus inhibiting RNA-protein interactions. N(6)-methyladenosine (m(6)A), the most abundant and dynamic internal modification in eukaryotic messenger RNA, can be selectively recognized by the YTHDF2 protein to affect the stability of cytoplasmic mRNAs, but how m(6)A achieves its wide-ranging physiological role needs further exploration. Here we show in human cells that m(6)A controls the RNA-structure-dependent accessibility of RBMs to affect RNA-protein interactions for biological regulation; we term this mechanism 'the m(6)A-switch'. We found that m(6)A alters the local structure in mRNA and long non-coding RNA (lncRNA) to facilitate binding of heterogeneous nuclear ribonucleoprotein C (HNRNPC), an abundant nuclear RNA-binding protein responsible for pre-mRNA processing. Combining photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) and anti-m(6)A immunoprecipitation (MeRIP) approaches enabled us to identify 39,060 m(6)A-switches among HNRNPC-binding sites; and global m(6)A reduction decreased HNRNPC binding at 2,798 high-confidence m(6)A-switches. We determined that these m(6)A-switch-regulated HNRNPC-binding activities affect the abundance as well as alternative splicing of target mRNAs, demonstrating the regulatory role of m(6)A-switches on gene expression and RNA maturation. Our results illustrate how RNA-binding proteins gain regulated access to their RBMs through m(6)A-dependent RNA structural remodelling, and provide a new direction for investigating RNA-modification-coded cellular biology.
| S-EPMC4355918 | biostudies-literature
Project description:Increasing awareness of the importance of protein-RNA interactions has motivated many approaches to predict residue-level RNA binding sites in proteins based on sequence or structural characteristics. Sequence-based predictors are usually high in sensitivity but low in specificity; conversely structure-based predictors tend to have high specificity, but lower sensitivity. Here we quantified the contribution of both sequence- and structure-based features as indicators of RNA-binding propensity using a machine-learning approach. In order to capture structural information for proteins without a known structure, we used homology modeling to extract the relevant structural features. Several novel and modified features enhanced the accuracy of residue-level RNA-binding propensity beyond what has been reported previously, including by meta-prediction servers. These features include: hidden Markov model-based evolutionary conservation, surface deformations based on the Laplacian norm formalism, and relative solvent accessibility partitioned into backbone and side chain contributions. We constructed a web server called aaRNA that implements the proposed method and demonstrate its use in identifying putative RNA binding sites.
| S-EPMC4150784 | biostudies-literature
Project description:The sequence of the DNA template has long been thought to influence the rate of transcription by DNA-dependent RNA polymerases, but the influence of DNA sequence on transcription elongation properties of eukaryotic RNA polymerase I (Pol I) from Saccharomyces cerevisiae has not been defined. In this study, we observe changes in dinucleotide production, transcription elongation complex stability, and Pol I pausing in vitro in response to downstream DNA. In vitro studies demonstrate that AT-rich downstream DNA enhances pausing by Pol I and inhibits Pol I nucleolytic cleavage activity. Analysis of Pol I native elongating transcript sequencing data in Saccharomyces cerevisiae suggests that these downstream sequence elements influence Pol I in vivo Native elongating transcript sequencing studies reveal that Pol I occupancy increases as downstream AT content increases and decreases as downstream GC content increases. Collectively, these data demonstrate that the downstream DNA sequence directly impacts the kinetics of transcription elongation prior to the sequence entering the active site of Pol I both in vivo and in vitro.
| S-EPMC6996881 | biostudies-literature
Project description:Evidence is accumulating that retroviruses can produce microRNAs (miRNAs). To prevent cleavage of their RNA genome, retroviruses have to use an alternative RNA source as miRNA precursor. The transacting responsive (TAR) hairpin structure in HIV-1 RNA has been suggested as source for miRNAs, but how these small RNAs are produced without impeding virus replication remained unclear. We used deep sequencing analysis of AGO2-bound HIV-1 RNAs to demonstrate that the 3' side of the TAR hairpin is processed into a miRNA-like small RNA. This ∼21 nt RNA product is able to repress the expression of mRNAs bearing a complementary target sequence. Analysis of the small RNAs produced by wild-type and mutant HIV-1 variants revealed that non-processive transcription from the HIV-1 LTR promoter results in the production of short TAR RNAs that serve as precursor. These TAR RNAs are cleaved by Dicer and processing is stimulated by the viral Tat protein. This biogenesis pathway differs from the canonical miRNA pathway and allows HIV-1 to produce the TAR-encoded miRNA-like molecule without cleavage of the RNA genome.
| S-EPMC4872094 | biostudies-literature