RNA最新文献

RNA-binding proteins shape biology through their widespread functions in RNA biochemistry. Their function requires the recognition of specific RNA motifs for targeted binding. These RNA-binding elements can be composed of both unmodified and chemically modified RNAs, of which over 170 chemical modifications have been identified in biology. Unmodified RNA sequence preferences for RNA-binding proteins have been widely studied, with numerous methods available to identify their preferred sequence motifs. However, only a few techniques can detect preferred RNA modifications, and no current method can comprehensively screen the vast array of hundreds of natural RNA modifications. Prior work demonstrated that λ-dynamics is an accurate in silico method to predict RNA base binding preferences of an RNA-binding antibody. This work extends that effort by using λ-dynamics to predict unmodified and modified RNA-binding preferences of human Pumilio, a prototypical RNA-binding protein. A library of RNA modifications was screened at eight nucleotide positions along the RNA to identify modifications predicted to affect Pumilio binding. Computed binding affinities were compared with experimental data to reveal high predictive accuracy. In silico force field accuracies were also evaluated between CHARMM36 and Amber RNA force fields to determine the best parameter set to use in binding calculations. This work demonstrates that λ-dynamics can predict RNA interactions to a bona fide RNA-binding protein without the requirements of chemical reagents or new methods to experimentally test binding at the bench. Advancing in silico methods like λ-dynamics will unlock new frontiers in understanding how RNA modifications shape RNA biochemistry.

In vertebrates, left-right (LR) asymmetry is specified by asymmetric decay of Dand5 messenger RNA (mRNA) mediated by the recruitment of the BicC family RNA binding protein 1 (Bicc1). Besides regulating organ laterality, Bicc1 is required to prevent cystic dilations in renal tubules and in pancreatic and bile ducts. However, validated target mRNAs are sparse in number, and how their binding to Bicc1 is regulated remains poorly understood. Bicc1 recruitment to Dand5 transcripts requires a conserved AGACGUGAC motif in the 3'UTR. Here, we report an N⁶-methyladenosine (m⁶A) in this sequence that disrupts binding to Bicc1 K homology (KH) domains in vitro, in stark contrast to IGF2BPs and FMR1, where m⁶A promotes RNA recognition by KH domains. We discuss the possible implications of this finding for LR axis formation and for a related role of Bicc1 in regulating specific target mRNAs in the kidney.

During the integrated stress response (ISR), most mRNAs exit translation and some condense into stress granules. Stress granules that form during chemotherapy can promote cancer cell survival and chemoresistance by an unknown mechanism. Cells can also spontaneously trigger the ISR at low levels, which promotes cellular quiescence where cells exit the cell cycle and are resistant to therapeutic agents. We hypothesized that the ability of cells to form stress granules might be a critical signal to drive cells into quiescence. Herein, we provide several observations that suggest stress granules enhance cell survival and chemoresistance by promoting cellular quiescence. The mechanism by which stress granules promote quiescence is by stimulating p21 expression, leading to inhibition of Rb phosphorylation. These results demonstrate that stress granule formation is sufficient to trigger cellular quiescence and argue that inhibitors of stress granules may be effective in combination with chemotherapy to limit the development of chemoresistance in treating human tumors.

Membrane-less organelles, dynamic subcellular structures formed by RNA and RNA-binding proteins (RBPs) undergoing liquid-liquid phase separation (LLPS), play key roles in biological processes such as RNA degradation in processing bodies (P-bodies), translation inhibition in stress granules, and RNA splicing in nuclear speckles. However, the study of RNA species within these organelles has been hindered by the absence of simple, sensitive, and specific methodologies. Here, we introduce target transcript amplification and sequencing (TATA-seq), a novel strategy for precisely profiling RNA in membrane-less organelles via in situ targeted transcription and linear amplification. TATA-seq uses a primary antibody against a marker protein of the target organelle to recruit a secondary antibody conjugated with streptavidin, which binds an oligonucleotide containing a T7 promoter. This initiates in situ RNA reverse transcription, followed by amplification with T7 RNA polymerase to generate sufficient material for sequencing, ensuring a duplication rate of no more than 25% and a mapping ratio of ∼90%. An IgG control is used to subtract background noise during data analysis. We demonstrate the method's utility by profiling RNA in stress granules induced by sodium arsenite in HeLa cells, with validation through FISH and immunofluorescence colocalization. TATA-seq offers a simple, highly sensitive, and accurate tool for studying RNA dynamics in membrane-less organelles, advancing the capabilities of RNA research.

PUF proteins (named for Drosophila melanogaster Pumilio and Caenorhabditis elegans fem-3 mRNA binding factor or FBF) are a family of RNA-binding proteins. C. elegans FBF is a collective term for two PUF proteins, FBF-1 and FBF-2, that maintain germline stem cells. FBF binds the 3'UTR of target RNAs and together with partner proteins represses translation of mRNAs that promote differentiation. Until recently, little was known about the functions of the FBF C-terminal intrinsically disordered regions that follow the RNA-binding domain (RBD). Despite high overall protein sequence conservation (91% identical residues), the FBF-1 and FBF-2 C-terminal tails (CTs) are distinct, and the FBF-2 CT is essential for its function. The FBF-2 CT contains a PUF-interacting motif (PIM) that binds its own RBD and autoinhibits RNA-binding affinity. Here we investigated whether differences in the FBF-1 and FBF-2 CTs impact molecular function. Unlike FBF-2, the FBF-1 CT had no impact on RNA binding. Despite this, a crystal structure of FBF-1 demonstrated that a PIM in the FBF-1 CT binds to its RBD, like FBF-2. By creating FBF-1/FBF-2 chimeric proteins, we discovered that the FBF-2 CT can autoinhibit FBF-1 RNA binding, and substitution of the FBF-1 PIM for the FBF-2 PIM diminished FBF-2 autoinhibition. Our results exemplify how RBP paralogs diverge to fine-tune their RNA-binding activities.

U7 snRNA is a 60 nucleotide component of U7 snRNP, a multisubunit endonuclease that cleaves precursors of metazoan replication-dependent histone mRNAs at the 3' end, hence generating mature histone mRNAs. The Sm site in U7 snRNA differs from the Sm site in spliceosomal snRNAs and promotes the assembly of a unique Sm ring containing Lsm10 and Lsm11 instead of the spliceosomal SmD1 and SmD2 proteins. While the spliceosomal-type Sm site is recognized by Gemin5, a subunit of the SMN complex, the identity of the protein that recognizes the unusual Sm site of U7 snRNA resulting in the incorporation of Lsm10 and Lsm11 has not been determined. Here, we looked for proteins in mammalian extracts that interact with U7 snRNA and identified polypyrimidine tract-binding protein 1 (PTBP1) and insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) as two major proteins with this characteristic. The binding of PTBP1 and IGF2BP3 to U7 snRNA depends on its unique Sm site and on the upstream CUCUUU motif that base-pairs with histone pre-mRNAs and defines substrate specificity of U7 snRNP. Among proteins that bind U7 snRNA, we also identified hnRNP A1. We show that hnRNP A1 interacts with the SMN protein of the SMN complex, a likely prerequisite for the protein that substitutes for Gemin5 in the assembly of U7-specific Sm ring. Our results also suggest a mechanism that explains why Gemin5 does not bind the Sm site of U7 snRNA.

The bacterial transcription termination factor Rho is a rare example of an RNA helicase that functions as a ring-shaped ATP-powered six-subunit motor. Recent studies have linked Rho's distinctive architecture to a variety of regulatory mechanisms that shape the bacterial transcriptome at the global scale and control the transcription of individual genes in a context-dependent manner. In this review, we provide a comprehensive overview of the molecular mechanisms by which Rho triggers transcription termination. We examine the two prevailing modes of Rho's action: the "catch-up" mode, where Rho actively translocates along RNA and collides with the RNA polymerase to terminate transcription, and the "stand-by" mode where Rho, recruited by transcription elongation factor NusG, remains poised to engage RNA polymerase at specific sites or under particular constraints. Additionally, we highlight Rho's interplay with nucleoid-structuring protein H-NS in the regulation of bacterial chromatin transcription, as well as the crucial role played by Rho in the conditional regulation of specific genomic loci. We discuss how these mechanisms contribute to the fine-tuning of gene activity and integrate into broader regulatory networks, supporting bacterial adaptation to environmental changes and resilience to external challenges.

Immortalized cell lines are commonly used as proxies for primary cells in human biology research. For example, Jurkat leukemic T cells fundamentally contributed to uncovering T-cell signaling, activation, and immune responses. However, the immortalization process can alter key cellular properties, and researchers widely believe this process could significantly change RNA modification machinery and modification sites. In this study, we focus on pseudouridine (ψ), one of the most abundant mRNA modifications, and compare ψ profiles in mRNA from primary and immortalized T cells using direct RNA sequencing (DRS). Surprisingly, 87% of ψ-sites were shared between the two cell types, primarily in transcripts encoding proteins involved in essential cellular processes, including RNA-modification regulation. Furthermore, the analysis of the 13% of sites unique to each cell type reveals that Jurkat cells contained transcripts linked to immune activation and oncogenesis, while primary T cells contained transcripts associated with calcium signaling and intracellular trafficking. We provide a list of these genes, which should be considered when using immortalized cells to study RNA modifications in immunology contexts. Most differences were driven by whether the mRNA was present or absent in the immortalized or primary cell type. Interestingly, RNA-modification enzyme expression levels were highly conserved in both cell types. This suggests that site-specific differences in ψ levels arise from regulatory processes acting in trans rather than differences in modification enzyme levels.

Detecting protein-coding genes in nucleotide sequences is a significant challenge for understanding genome and transcriptome function, yet the reliability of bioinformatic tools for this task remains largely unverified. This is despite some tools being available for several decades and widely used for genome and transcriptome annotation. We perform an assessment of nucleotide sequence and alignment-based de novo protein-coding detection tools. The controls we use exclude any previous training data set and include coding exons as a positive set and length-matched intergenic and shuffled sequences as negative sets. Our work demonstrates that several widely used tools are neither accurate nor computationally efficient for the protein-coding sequence detection problem. In fact, just three of nine tools significantly outperformed a naive scoring scheme. Furthermore, we note a high discrepancy between self-reported accuracies and the accuracy achieved in our study. Our results show that the extra dimension from conserved and variable nucleotides in alignments has a significant advantage over single-sequence approaches. These results highlight significant limitations in existing protein-coding annotation tools that are widely used for lncRNA annotation. This shows a need for more robust and efficient approaches to training and assessing the performance of tools for identifying protein-coding sequences. Our study paves the way for future advancements in comparative genomic approaches, and we hope will popularize more robust approaches to genome and transcriptome annotation.