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Methylated cytosines in total poly(A) ESC RNA. (XLSX 761 kb

Synthesis, Thermodynamic Properties, and Crystal Structure of RNA Oligonucleotides Containing 5‑Hydroxymethylcytosine

5-Hydroxymethylcytosine (hm⁵C) is an RNA modification that has attracted significant interest because of the finding that RNA hydroxymethylation can favor mRNA translation. For insight into the mechanistic details of hm⁵C function to be obtained, the availability of RNAs containing this modification at defined positions that can be used for in vitro studies is highly desirable. In this work, we present an eight-step route to 5-hydroxymethylcytidine (hm⁵rC) phosphoramidite for solid-phase synthesis of modified RNA oligonucleotides. Furthermore, we examined the effects of hm⁵rC on RNA duplex stability and its impact on structure formation using the sarcin-ricin loop (SRL) motif. Thermal denaturation experiments revealed that hm⁵rC increases RNA duplex stability. By contrast, when cytosine within an UNCG tetraloop motif was replaced by hm⁵rC, the thermodynamic stability of the corresponding hairpin fold was attenuated. Importantly, incorporation of hm⁵rC into the SRL motif resulted in an RNA crystal structure at 0.85 Å resolution. Besides changes in the hydration pattern at the site of modification, a slight opening of the hm⁵rC–G pair compared to the unmodified C–G in the native structure was revealed

Additional file 1: Table S1. of Distinct 5-methylcytosine profiles in poly(A) RNA from mouse embryonic stem cells and brain

General sequencing and m5C calling results. (XLSX 34 kb

Additional file 10: Table S9. of Distinct 5-methylcytosine profiles in poly(A) RNA from mouse embryonic stem cells and brain

Primer sequences. (XLSX 25 kb

Additional file 4: Table S3. of Distinct 5-methylcytosine profiles in poly(A) RNA from mouse embryonic stem cells and brain

Methylated cytosines in total poly(A) ESC RNA. (XLSX 761 kb

Structural and Biochemical Characterization of the Bilin Lyase CpcS from Thermosynechococcus elongatus

Cyanobacterial phycobiliproteins have evolved to capture light energy over most of the visible spectrum due to their bilin chromophores, which are linear tetrapyrroles that have been covalently attached by enzymes called bilin lyases. We report here the crystal structure of a bilin lyase of the CpcS family from Thermosynechococcus elongatus (<i>Te</i>CpcS-III). <i>Te</i>CpcS-III is a 10-stranded β barrel with two alpha helices and belongs to the lipocalin structural family. <i>Te</i>CpcS-III catalyzes both cognate as well as noncognate bilin attachment to a variety of phycobiliprotein subunits. <i>Te</i>CpcS-III ligates phycocyanobilin, phycoerythrobilin, and phytochromobilin to the alpha and beta subunits of allophycocyanin and to the beta subunit of phycocyanin at the Cys82-equivalent position in all cases. The active form of <i>Te</i>CpcS-III is a dimer, which is consistent with the structure observed in the crystal. With the use of the UnaG protein and its association with bilirubin as a guide, a model for the association between the native substrate, phycocyanobilin, and <i>Te</i>CpcS was produced