Bechara, E. | Search bioRxiv

bioRxiv2023

Internal Ribosome Entry Sites act as Effector Domain in linear and circular antisense long non-coding SINEUP RNAs

SINEUPs are antisense long non-coding RNAs that enhance translation of overlapping sense mRNAs through the activity of two domains: a SINEB2 sequence UP-regulating translation (Effector Domain, ED) and an antisense region providing target specificity (Binding Domain, BD). In this study, we demonstrate that the invSINEB2 sequence from the natural SINEUP AS Uchl1 RNA is an Internal Ribosomal Entry Site (IRES) when acting in cis and that known viral and cellular IRES sequences can act as Effector Domain in synthetic SINEUPs. To identify natural IRES-containing, non-coding RNAs with SINEUP-like activity, we focused on circular RNAs showing that the non-coding circ5533, transcribed from the c-myc locus, enhances endogenous protein expression of its target PX Domain Containing Serine/Threonine Kinase Like (Pxk) by increasing mRNA association to polysomes. In summary, this study shows that natural and synthetic SINEUPs include linear and circular transcripts with an embedded IRES sequence as ED.

molecular biology↗

bioRxiv2022

The RNA-Binding Protein SAM68 regulates cardiomyocyte differentiation by enhancing Gata4 translation

The signal transduction and activation of RNA (STAR) family is composed of RNA-binding proteins (RBPs) that play a central role in mammalian development. Nonetheless, the functions and modes of action that STAR proteins have in lineage specification are still poorly understood. Here, we characterized the role of STAR proteins SAM68 and QUAKING (QKI) in pluripotency and differentiation by performing their depletion through CRISPR-Cas9 in mouse embryonic stem cells (mESCs). Combining RNA-sequencing, ribosome profiling and advanced computational predictions, we found that both SAM68 and QKI regulate the mESCs self-renewal and are indispensable for cardiomyocyte differentiation. At the molecular level, we discovered that SAM68 and QKI antagonistically control the expression of cardiogenic factors. Our calculations indicated that SAM68, unlike QKI, binds the cardiogenic-specific transcription factor Gata4 in a region spanning nucleotides 500 to 1000 of the mRNA corresponding to part of the 5 untranslated region and the first exon. We validated the predictions by electrophoretic mobility shift assay and RNA immunoprecipitation showing that SAM68 controls the translation of Gata4 during mESCs differentiation towards the cardiomyocyte lineage.

molecular biology↗