Hansen, S. R. | Search bioRxiv

bioRxiv2021

Miniature Structured Illumination Microscope for in vivo 3D Imaging of Brain Structures with Optical Sectioning

We present a high-resolution miniature, light-weight fluorescence microscope with electrowetting lens and onboard CMOS for high resolution volumetric imaging and structured illumination for rejection of out-of-focus and scattered light. The miniature microscope (SIMscope3D) delivers structured light using a coherent fiber bundle to obtain optical sectioning with an axial resolution of 18 m. Volumetric imaging of eGFP labeled cells in fixed mouse brain tissue at depths up to 220 m is demonstrated. The functionality of SIMscope3D to provide background free 3D imaging is shown by recording time series of microglia dynamics in awake mice at depths up to 120 m in the brain.

bioengineering↗

bioRxiv2021

Multi-Factor Authentication of Potential 5' Splice Sites by the Saccharomyces cerevisiae U1 snRNP

In eukaryotes, splice sites define the introns of pre-mRNAs and must be recognized and excised with nucleotide precision by the spliceosome to make the correct mRNA product. In one of the earliest steps of spliceosome assembly, the U1 small nuclear ribonucleoprotein (snRNP) recognizes the 5 splice site (5 SS) through a combination of base pairing, protein-RNA contacts, and interactions with other splicing factors. Previous studies investigating the mechanisms of 5 SS recognition have largely been done in vivo or in cellular extracts where the U1/5 SS interaction is difficult to deconvolute from the effects of trans-acting factors or RNA structure. In this work we used co-localization single-molecule spectroscopy (CoSMoS) to elucidate the pathway of 5 SS selection by purified yeast U1 snRNP. We determined that U1 reversibly selects 5 SS in a sequence-dependent, two-step mechanism. A kinetic selection scheme enforces pairing at particular positions rather than overall duplex stability to achieve long-lived U1 binding. Our results provide a kinetic basis for how U1 may rapidly surveil nascent transcripts for 5 SS and preferentially accumulate at these sequences rather than on close cognates. IMPACT STATEMENTThe yeast U1 snRNP recognizes multiple features of target RNAs to reversibly identify splicing-competent 5 splice sites.

biochemistry↗