No abstract

The heptad repeats of the C-terminal domain (CTD) of RNA polymerase II (Pol II) are extensively modified throughout the transcription cycle. The CTD coordinates RNA synthesis and processing by recruiting transcription regulators as well as RNA capping, splicing and 3’end processing factors. The SPOC domain of PHF3 was recently identified as a CTD reader domain specifically binding to phosphorylated serine-2 residues in adjacent CTD repeats. Here, we establish the SPOC domains of the human proteins DIDO, SHARP (also known as SPEN) and RBM15 as phosphoserine binding modules that can act as CTD readers but also recognize other phosphorylated binding partners. We report the crystal structure of SHARP SPOC in complex with CTD and identify the molecular determinants for its specific binding to phosphorylated serine-5. PHF3 and DIDO SPOC domains preferentially interact with the Pol II elongation complex, while RBM15 and SHARP SPOC domains engage with writers and readers of m6A, the most abundant RNA modification. RBM15 positively regulates m6A levels and mRNA stability in a SPOC-dependent manner, while SHARP SPOC is essential for its localization to inactive X-chromosomes. Our findings suggest that the SPOC domain is a major interface between the transcription machinery and regulators of transcription and co-transcriptional processes.

No abstract

In this report, we perform structure validation of recently reported RNA phosphorothioate (PT) modifications, a new set of epitranscriptome marks found in bacteria and eukaryotes including humans. By comparing synthetic PT‐containing diribonucleotides with native species in RNA hydrolysates by high‐resolution mass spectrometry (MS), metabolic stable isotope labeling, and PT‐specific iodine‐desulfurization, we disprove the existence of PTs in RNA from E. coli, S. cerevisiae, human cell lines, and mouse brain. Furthermore, we discuss how an MS artifact led to the initial misidentification of 2′‐O‐methylated diribonucleotides as RNA phosphorothioates. To aid structure validation of new nucleic acid modifications, we present a detailed guideline for MS analysis of RNA hydrolysates, emphasizing how the chosen RNA hydrolysis protocol can be a decisive factor in discovering and quantifying RNA modifications in biological samples.

Backgrounded membrane imaging (BMI) is a novel automated, 96-well plate-based microscopic approach for subvisible particle analysis. We scientifically evaluated BMI with respect to sizing and counting accuracy, working range, impact of refractive index, and interferences by silicone oil droplets, and compared BMI to state-of-the-art dynamic image analysis (DIA). Image quality was found to be comparable to current DIA methodologies. However, with the first versions of BMI image analysis software, an undersizing of polystyrene beads was observed. BMI linear concentration range was found to reach an upper limit (7.1 Â 10 5 particles/mL) similar to DIA. In the absence of silicone oil droplets, BMI and DIA showed good agreement in total particle concentrations (particle diameter !2 mm) but differences in size distributions for particle sizes !4 mm. Analyses of prefilled syringe products and silicone oil emulsions demonstrated the removal of silicone oil in BMI sample processing. In contrast to DIA, particle counting by BMI remained unaffected by changes in refractive index. Overall, we demonstrated BMI to be a promising orthogonal method for subvisible particle characterization. Aspects like low required sample volume, high throughput, and ease of handling can make BMI a valuable alternative or complement to DIA in particular for formulation screening.