Insights into the role of methionine synthase in the universal C-13 depletion in O- and N-methyl groups of natural products

International audienceMany O-methyl and N-methyl groups in natural products are depleted in C-13 relative to the rest of the molecule. These methyl groups are derived from the C-1 tetrahydrofolate pool via L-methionine, the principle donor of methyl units. Depletion could occur at a number of steps in the pathway. We have tested the hypothesis that methionine biosynthesis is implicated in this depletion by using a combined experimental and theoretical approach. By using isotope ratio monitoring C-13 NMR spectrometry to measure the position-specific distribution of C-13 within L-methionine of natural origin, it is shown that the S-methyl group is depleted in C-13 by similar to 20%o relative to the other positions in the molecule. In parallel, we have conducted a basic theoretical analysis of the reaction pathway of methionine synthase to assess whether the enzyme cobalamin-independent L-methionine synthase (EC 2.1.1.14) that catalyzes the synthesis of L-methionine from 5-methyl-tetrahydrofolate and homocysteine plays a role in causing this depletion. Calculation predicts a strong normal C-13 kinetic isotope effect (1.087) associated with this enzyme. Hence, depletion in C-13 in the S-methyl of L-methionine during biosynthesis can be identified as an important factor contributing to the general depletion seen in many O methyl and N-methyl groups of natural products

Post-transcriptional modifications contribuye to the overexpression of cyclin D2 in multiple myeloma

[Purpose]: Dysregulation of one of the three D-cyclin genes has been observed in virtually all multiple myeloma tumors. The mechanisms by which CCND2 is upregulated in a set of multiple myeloma are not completely deciphered. We investigated the role of post-transcriptional regulation through the interaction between miRNAs and their binding sites at 3′UTR in CCND2 overexpression in multiple myeloma. [Experimental Design]: Eleven myeloma cell lines and 45 primary myeloma samples were included in the study. Interactions between miRNAs deregulated in multiple myeloma and mRNA targets were analyzed by 3′UTR-luciferase plasmid assay. The presence of CCND2 mRNA isoforms different in length was explored using qRT-PCR, Northern blot, mRNA FISH, and 3′ rapid amplification of cDNA ends (RACE)-PCR. [Results]: We detected the presence of short CCND2 mRNA, both in the multiple myeloma cell lines and primary cells. The results obtained by 3′RACE experiments revealed that changes in CCND2 3′UTR length are explained by alternative polyadenylation. The luciferase assays using plasmids harboring the truncated CCND2 mRNA strongly confirmed the loss of miRNA sites in the shorter CCND2 mRNA isoform. Those multiple myelomas with greater abundance of the shorter 3′UTR isoform were associated with significant higher level of total CCND2 mRNA expression. Furthermore, functional analysis showed significant CCND2 mRNA shortening after CCND1 silencing and an increased relative expression of longer isoform after CCND1 and CCND3 overexpression, suggesting that cyclin D1 and D3 could regulate CCND2 levels through modifications in polyadenylation-cleavage reaction. [Conclusions]: Overall, these results highlight the impact of CCND2 3′UTR shortening on miRNA-dependent regulation of CCND2 in multiple myeloma.This study was partially supported by Instituto de Salud Carlos III (Fondo de Investigaciones Sanitarias: PI08/0568 and PI13/00111), Asociacion Española Contra el Cancer (AECC, GCB120981SAN), >Gerencia Regional de Salud, Junta de Castilla y Leon> (GRS 702/A/11, BIO/SA57/13 and BIO/SA35/14) grants, the Spanish Myeloma Network Program (RD12/0036/0058) and the INNOCAMPUS Program (CEI10-1-0010). I. Misiewicz-Krzeminska was supported by Instituto de Salud Carlos III (PS09/01897). M.E. Sarasquete was supported by Contrato Miguel Servet (CP13/00080). Research in the I. Sanchez-García group was supported by FEDER and MINECO (SAF2012-32810, and Red de Excelencia Consolider OncoBIO SAF2014-57791-REDC), Instituto de Salud Carlos III (PIE14/00066), NIH (R01 CA109335-04A1), >Junta de Castilla y Leon> (BIO/SA32/14 and CSI001U14) grants, the Fundacion Inocente Inocente, the German Carreras Foundation (DJCLS R13/26) and by the ARIMMORA project (European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 282891). Research in the C.V. Duenas group was supported by >Miguel Servet> Grant (CP14/00082 AES 2013-2016) from the Instituto de Salud Carlos III (Ministerio de Economía y Competitividad). I. Sanchez-García is an API lab of the EuroSyStem project and a partner within the Marie Curie Initial Training Network DECIDE.Peer Reviewe