Ataxin-2 (Atx2), an RNA-binding protein, plays a pivotal role in the regulation of RNA, intracellular metabolism, and translation within the cellular environment. Although both the Sm-like (LSm) and LSm-associated (LSmAD) domains are considered to associated with RNA binding, there is still a lack of experimental evidence supporting their functions. To address this, we designed and constructed several recombinants containing the RNA-binding domain (RBD) of Atx2. By employing biophysical and biochemical techniques, such as EMSA and SHAPE chemical detection, we identified that LSm is responsible for RNA binding, whereas LSmAD alone does not bind RNA. NMR and small-angle X-ray scattering (SAXS) analyses have revealed that the LSmAD domain exhibits limited structural integrity and poor folding capability. The EMSA data confirmed that both LSm and LSm-LSmAD bind RNA, whereas LSmAD alone cannot, suggesting that LSmAD may serve as an auxiliary role to the LSm domain. SHAPE chemical probing further demonstrates that LSm binds to the AU-rich, GU-rich, or CU-rich sequence, but not to the CA-rich sequence. These findings indicate that Atx2 can interact with the U-rich sequences in the 3′-UTR, implicating its role in poly(A) tailing and the regulation of mRNA translation and degradation.

Riboswitches are regulatory noncoding RNAs found in bacteria, fungi and plants, that modulate gene expressions through structural changes in response to ligand binding. Understanding how ligands interact with riboswitches in solution can shed light on the molecular mechanisms of this ancient regulators. Previous studies showed that riboswitches undergo global conformation changes in response to ligand binding to relay information. Here, we report conformation switching models of the recently discovered tetrahydrofolic acid-responsive second class of tetrahydrofolate (THF-II) riboswitches in response to ligand binding. Using a combination of selective 2′-hydroxyl acylation, analyzed by primer extension (SHAPE) assay, 3D modeling and small-angle X-ray scattering (SAXS), we found that the ligand specifically recognizes and reshapes the THF-II riboswitch loop regions, but does not affect the stability of the P3 helix. Our results show that the THF-II riboswitch undergoes only local conformation changes in response to ligand binding, rearranging the Loop1-P3-Loop2 region and rotating Loop1 from a ~120° angle to a ~75° angle. This distinct conformation changes suggest a unique regulatory mechanism of the THF-II riboswitch, previously unseen in other riboswitches. Our findings may contribute to the fields of RNA sensors and drug design.

Circular RNA (circRNA) has emerged as a promising vector for drug delivery because, unlike linear mRNA, it does not require costly chemical modifications and offers greater stability and sustained expression in cells. Lacking the canonical 5′ cap structure, circRNA relies primarily on internal ribosome entry sites (IRES) to initiate translation, but IRES-mediated initiation is less efficient than cap-dependent translation. To overcome this limitation, we devised a dual-IRES strategy that introduces a second IRES to drive translation of the coding sequence (CDS). By testing several IRES elements known for high translational activity, this study shows that IRESs derived from the EMCV (Encephalomyocarditis virus) family can enhance expression when placed at the 3′ of the CDS, in coordination with the 5′ EMCV-derived IRES. The optimal dual-IRES combinations identified in this study display compatibility with two different coding sequences, offering a useful strategy to enhance circRNA translation.

The Bifidobacterium bifidum SAM-VI riboswitch undergoes dynamic conformational changes that modulate downstream gene expression. Traditional structural methods such as crystallography capture the bound conformation at high resolution, and additional efforts would reveal details from the dynamic transition. Here, we revealed a transcription-dependent conformation model for Bifidobacterium bifidum SAM-VI riboswitch. In this study, we combine small-angle X-ray scattering, chemical probing, and isothermal titration calorimetry to unveil the ligand-binding properties and conformational changes of the Bifidobacterium bifidum SAM-VI riboswitch and its variants. Our results suggest that the SAM-VI riboswitch contains a pre-organized ligand-binding pocket and stabilizes into the bound conformation upon binding to SAM. Whether the P1 stem formed and variations in length critically influence the conformational dynamics of the SAM-VI riboswitch. Our study provides the basis for artificially engineering the riboswitch by manipulating its peripheral sequences without modifying the SAM-binding core.

The success of messenger RNA (mRNA) vaccines in controlling COVID-19 has warranted further developments in new technology. Currently, their quality control process largely relies on low-resolution electrophoresis for detecting chain breaks. Here, we present an approach using multi-primer reverse transcription sequencing (MPRT-seq) to identify degradation fragments in mRNA products. Using this in-house-made mRNA containing two antigens and untranslated regions (UTRs), we analyzed the mRNA completeness and degradation pattern at a nucleotide resolution. We then analyzed the sensitive base sequence and its correlation with the secondary structure. Our MPRT-seq mapping shows that certain sequences on the 5′ of bulge–stem–loop structures can result in preferential chain breaks. Our results agree with commonly used capillary electrophoresis (CE) integrity analysis but at a much higher resolution, and can improve mRNA stability by providing information to remove sensitive structures or sequences in the mRNA sequence design.