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Translational control is crucial for well-balanced cellular function and viability of organisms. Different mechanisms have evolved to up- and down-regulate protein synthesis, including 3' untranslated region (UTR)-mediated translation repression. RNA binding proteins or microRNAs interact with regulatory sequence elements located in the 3' UTR and interfere most often with the rate-limiting initiation step of translation. Dysregulation of post-transcriptional gene expression leads to various kinds of diseases, emphasizing the significance of understanding the mechanisms of these processes. So far, only limited mechanistic details about kinetics and dynamics of translation regulation are understood. This mini-review focuses on 3' UTR-mediated translational regulation mechanisms and demonstrates the potential of using single-molecule fluorescence-microscopy for kinetic and dynamic studies of translation regulation in vivo and in vitro. [ABSTRACT FROM AUTHOR]

Cellular protein-RNA complexes assemble on nascent transcripts, but methods to observe transcription and protein binding in real time and at physiological concentrations are not available. Here, we report a single-molecule approach based on zero-mode waveguides that simultaneously tracks transcription progress and the binding of ribosomal protein S15 to nascent RNA transcripts during early ribosome biogenesis. We observe stable binding of S15 to single RNAs immediately after transcription for the majority of the transcripts at 35 °C but for less than half at 20 °C. The remaining transcripts exhibit either rapid and transient binding or are unable to bind S15, likely due to RNA misfolding. Our work establishes the foundation for studying transcription and its coupled co-transcriptional processes, including RNA folding, ligand binding, and enzymatic activity such as in coupling of transcription to splicing, ribosome assembly or translation.

Author(s): Olivier Duss [sup.1] , Erich Michel [sup.1] , Maxim Yulikov [sup.2] , Mario Schubert [sup.1] , Gunnar Jeschke [sup.2] , Frédéric H.-T. Allain [sup.1] Author Affiliations: (1) Institute of [...], MicroRNA and protein sequestration by non-coding RNAs (ncRNAs) has recently generated much interest. In the bacterial Csr/Rsm system, which is considered to be the most general global post-transcriptional regulatory system responsible for bacterial virulence, ncRNAs such as CsrB or RsmZ activate translation initiation by sequestering homodimeric CsrA-type proteins from the ribosome-binding site of a subset of messenger RNAs. However, the mechanism of ncRNA-mediated protein sequestration is not understood at the molecular level. Here we show for Pseudomonas fluorescens that RsmE protein dimers assemble sequentially, specifically and cooperatively onto the ncRNA RsmZ within a narrow affinity range. This assembly yields two different native ribonucleoprotein structures. Using a powerful combination of nuclear magnetic resonance and electron paramagnetic resonance spectroscopy we elucidate these 70-kilodalton solution structures, thereby revealing the molecular mechanism of the sequestration process and how RsmE binding protects the ncRNA from RNase E degradation. Overall, our findings suggest that RsmZ is well-tuned to sequester, store and release RsmE and therefore can be viewed as an ideal protein 'sponge'. A novel combined NMR and EPR spectroscopy approach reveals the structure and assembly mechanism of a 70-kDa bacterial ribonucleoprotein complex acting as a protein sponge in translational regulation. RsmZ, a small RNA with an appetite for protein Bacterial virulence can involve a general system of post-transcriptional regulation by non-coding RNAs such as RsmZ that sequester inhibitory proteins (commonly the CsrA-type protein RsmE) thereby freeing their target mRNAs and promoting translation activation. Using NMR and pulsed-electron paramagnetic resonance spectroscopy, Frédéric Allain and colleagues have determined how RsmE dimers assemble on RsmZ to form a 70 kD ribonucleoprotein particle and protect it from degradation. Their data indicate how RsmZ sequesters, stores and releases proteins, acting as a potent protein 'sponge'.

The cyclooxygenase-2 is a pro-inflammatory and cancer marker, whose mRNA stability and translation is regulated by the CUG-binding protein 2 interacting with AU-rich sequences in the 3' untranslated region. Here, we present the solution NMR structure of CUG-binding protein 2 RRM3 in complex with 5'-UUUAA-3' originating from the COX-2 3'-UTR. We show that RRM3 uses the same binding surface and protein moieties to interact with AU- and UG-rich RNA motifs, binding with low and high affinity, respectively. Using NMR spectroscopy, isothermal titration calorimetry and molecular dynamics simulations, we demonstrate that distinct sub-states characterized by different aromatic side-chain conformations at the RNA-binding surface allow for high- or low-affinity binding with functional implications. This study highlights a mechanism for RNA discrimination possibly common to multiple RRMs as several prominent members display a similar rearrangement of aromatic residues upon binding their targets. [ABSTRACT FROM AUTHOR]

Protein-RNA complexes exist in many forms within the cell, from stable machines such as the ribosome to transient assemblies like the spliceosome. All protein-RNA assemblies rely on spatially and temporally coordinated interactions between specific proteins and RNAs to achieve a functional form. RNA folding and structure are often critical for successful protein binding and protein-RNA complex formation. RNA modifications change the chemical nature of a given RNA and often alter its folding kinetics. Both these alterations can affect how and if proteins or other RNAs can interact with the modified RNA and assemble into complexes. N 6 -methyladenosine (m 6 A) is the most common base modification on mRNAs and regulatory noncoding RNAs and has been shown to impact RNA structure and directly modulate protein-RNA interactions. In this review, focusing on the mechanisms and available quantitative information, we discuss first how the METTL3/14 m 6 A writer complex is specifically targeted to RNA assisted by protein-RNA and other interactions to enable site-specific and co-transcriptional RNA modification and, once introduced, how the m 6 A modification affects RNA folding and protein-RNA interactions., (© 2023 Höfler and Duss.)

Posttranscriptional regulation of gene expression by small noncoding RNAs (sRNAs) is an important control mechanism that modulates bacterial metabolism, motility, and pathogenesis. Using the bacterial carbon storage regulator/regulator of secondary metabolism (Csr/Rsm) system, we here describe an E. coli-based cell-free translation assay that allows a quantitative analysis of translation regulation by ncRNAs and their corresponding translation repressor proteins. The assay quantifies the translation of chloramphenicol acetyltransferase in cell-free expression reactions that contain defined amounts of ncRNA and repressor protein. We demonstrate our protocol with a comparative translation activation analysis of the RsmX, RsmY, and RsmZ sRNAs from Pseudomonas protegens, which reveals a superior efficacy of RsmZ over RsmX and RsmY.