Discrimination between Closely Related Cellular Metabolites by the SAM-I Riboswitch

The SAM-I riboswitch is a cis-acting element of genetic control found in bacterial mRNAs that specifically binds S-adenosylmethionine (SAM). We previously determined the 2.9-Å X-ray crystal structure of the effector-binding domain of this RNA element, revealing details of RNA-ligand recognition. To improve this structure, variations were made to the RNA sequence to alter lattice contacts, resulting in a 0.5-Å improvement in crystallographic resolution and allowing for a more accurate refinement of the crystallographic model. The basis for SAM specificity was addressed by a structural analysis of the RNA complexed to S-adenosylhomocysteine (SAH) and sinefungin and by measuring the affinity of SAM and SAH for a series of mutants using isothermal titration calorimetry. These data illustrate the importance of two universally conserved base pairs in the RNA that form electrostatic interactions with the positively charged sulfonium group of SAM, thereby providing a basis for discrimination between SAM and SAH.     

The S(MK) box is a new SAM-binding RNA for translational regulation of SAM synthetase

We have identified the S(MK) box as a conserved RNA motif in the 5' untranslated leader region of metK (SAM synthetase) genes in lactic acid bacteria, including Enterococcus, Streptococcus and Lactococcus species. This RNA element bound SAM in vitro, and binding of SAM caused an RNA structural rearrangement that resulted in sequestration of the Shine-Dalgarno (SD) sequence. Mutations that disrupted pairing between the SD region and a sequence complementary to the SD blocked SAM binding, whereas compensatory mutations that restored pairing restored SAM binding. The Enterococcus faecalis S(MK) box conferred translational repression of a lacZ reporter when cells were grown under conditions where SAM pools are elevated, and mutations that blocked SAM binding resulted in loss of repression, demonstrating that the S(MK) box is functional in vivo. The S(MK) box therefore represents a new SAM-binding riboswitch distinct from the previously identified S box RNAs.     

Structural characterization reveals that viperin is a radical S-adenosyl-l-methionine (SAM) enzyme

Viperin is an interferon-inducible protein inhibiting many DNA and RNA viruses. It contains an N-terminal transmembrane helix, a highly conserved C-terminus and a middle region carrying a CX3CX2C motif, characteristic of radical S-adenosyl-l-methionine (SAM) enzymes. So far no structural characterization has been reported and reconstitution of the [4Fe-4S] cluster in viperin all failed. Here, by dissecting the 361-residue human viperin into 12 fragments, followed by extensive CD and NMR characterization, Viperin (45-361) was identified to be soluble and structured in buffers. Most importantly, we have successfully reconstituted the [4Fe-4S] cluster in Viperin (45-361), thus providing the first experimental evidence confirming that viperin is indeed a radical SAM enzyme. Furthermore, the C-terminus Viperin (214-361) which is insoluble in buffers but again can be solubilized in salt-free water appears to be only partially folded. Our results thus imply that the radical SAM enzyme activity may play a key role in the broad antiviral actions of viperin.     

HL-60细胞内DNA甲基化作用与RNA聚合酶活力的关系

以 S-腺苷酰 - L-甲硫氨酸 ( SAM)为诱导物 ,在 1 0 μmol/L最佳浓度下可诱导 HL- 60细胞分化达 1 6%左右 .HPLC测定结果证明 ,诱导物处理后 HL- 60细胞 DNA甲基化水平升高 .通过 3 H-UTP同位素参入法 ,测定了不同处理时间和不同浓度 SAM对 HL- 60细胞 DNA模板体外转录活性的影响 ,发现体外活力下降 .比较了不同浓度α-鹅膏蕈碱存在下 RNA聚合酶活力的变化 ,结果表明 SAM处理后细胞中不同 RNA转录产物所占份额改变     

West Nile virus methyltransferase catalyzes two methylations of the viral RNA cap through a substrate-repositioning mechanism

Flaviviruses encode a single methyltransferase domain that sequentially catalyzes two methylations of the viral RNA cap, GpppA-RNA-->m(7)GpppA-RNA-->m(7)GpppAm-RNA, by using S-adenosyl-l-methionine (SAM) as a methyl donor. Crystal structures of flavivirus methyltransferases exhibit distinct binding sites for SAM, GTP, and RNA molecules. Biochemical analysis of West Nile virus methyltransferase shows that the single SAM-binding site donates methyl groups to both N7 and 2'-O positions of the viral RNA cap, the GTP-binding pocket functions only during the 2'-O methylation, and two distinct sets of amino acids in the RNA-binding site are required for the N7 and 2'-O methylations. These results demonstrate that flavivirus methyltransferase catalyzes two cap methylations through a substrate-repositioning mechanism. In this mechanism, guanine N7 of substrate GpppA-RNA is first positioned to SAM to generate m(7)GpppA-RNA, after which the m(7)G moiety is repositioned to the GTP-binding pocket to register the 2'-OH of the adenosine with SAM, generating m(7)GpppAm-RNA. Because N7 cap methylation is essential for viral replication, inhibitors designed to block the pocket identified for the N7 cap methylation could be developed for flavivirus therapy.     

Solution structure of the Vts1 SAM domain in the presence of RNA

The yeast Vts1 SAM (sterile alpha motif) domain is a member of a new class of SAM domains that specifically bind RNA. To elucidate the structural basis for RNA binding, the solution structure of the Vts1 SAM domain, in the presence of a specific target RNA, has been solved by multidimensional heteronuclear NMR spectroscopy. The Vts1 SAM domain retains the "core" five-helix-bundle architecture of traditional SAM domains, but has additional short helices at N and C termini, comprising a small substructure that caps the core helices. The RNA-binding surface of Vts1, determined by chemical shift perturbation, maps near the ends of three of the core helices, in agreement with mutational data and the electrostatic properties of the molecule. These results provide a structural basis for the versatility of the SAM domain in protein and RNA-recognition.     

Uptake and utilization of S-adenosyl-L-methionine and S-adenosyl-L-homocysteine in an adenine mutant of Saccharomyces cerevisiae

An adenine mutant of Saccharomyces cerevisiae was able to utilize S-adenosyl-l-methionine (SAM), S-adenosyl-l-homocysteine (SAH), or adenine as sources for growth and ribonucleic acid (RNA) synthesis. Exogenous SAM or SAH was degraded after entering the cell, and the adenine moiety was reutilized in the endogenous synthesis of the thionium compounds. Part of this endogenous synthesis proceeds from an undetermined de novo system of purine synthesis which contributes significantly to the synthesis of SAM and less so to the RNA purines. Some of the methyl groups of exogenous SAM-methyl-(3)H were incorporated into ribosomal and transfer RNA. Methionine partially antagonized this incorporation. The uptake of SAM-adenine-8-(14)C was not affected by the presence of equal quantities of SAH or adenine in the medium. Exogenous SAM affected the uptake of exogenous SAH-adenine-8-(14)C or adenine-8-(14)C. Exogenous SAH inhibited the uptake of exogenous adenine-8-(14)C.     

Common themes and differences in SAM recognition among SAM riboswitches

The recent discovery of short cis-acting RNA elements termed riboswitches has caused a paradigm shift in our understanding of genetic regulatory mechanisms. The three distinct superfamilies of S-adenosyl-l-methionine (SAM) riboswitches are the most commonly found riboswitch classes in nature. These RNAs represent three independent evolutionary solutions to achieve specific SAM recognition. This review summarizes research on 1) modes of gene regulatory mechanisms, 2) common themes and differences in ligand recognition, and 3) ligand-induced conformational dynamics among SAM riboswitch families. The body of work on the SAM riboswitch families constitutes a useful primer to the topic of gene regulatory RNAs as a whole.     

mTORC1-independent translation control in mammalian cells by methionine adenosyltransferase 2A and S-adenosylmethionine

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (SAM). As the sole methyl-donor for methylation of DNA, RNA, and proteins, SAM levels affect gene expression by changing methylation patterns. Expression of MAT2A, the catalytic subunit of isozyme MAT2, is positively correlated with proliferation of cancer cells; however, how MAT2A promotes cell proliferation is largely unknown. Given that the protein synthesis is induced in proliferating cells and that RNA and protein components of translation machinery are methylated, we tested here whether MAT2 and SAM are coupled with protein synthesis. By measuring ongoing protein translation via puromycin labeling, we revealed that MAT2A depletion or chemical inhibition reduced protein synthesis in HeLa and Hepa1 cells. Furthermore, overexpression of MAT2A enhanced protein synthesis, indicating that SAM is limiting under normal culture conditions. In addition, MAT2 inhibition did not accompany reduction in mechanistic target of rapamycin complex 1 activity but nevertheless reduced polysome formation. Polysome-bound RNA sequencing revealed that MAT2 inhibition decreased translation efficiency of some fraction of mRNAs. MAT2A was also found to interact with the proteins involved in rRNA processing and ribosome biogenesis; depletion or inhibition of MAT2 reduced 18S rRNA processing. Finally, quantitative mass spectrometry revealed that some translation factors were dynamically methylated in response to the activity of MAT2A. These observations suggest that cells possess an mTOR-independent regulatory mechanism that tunes translation in response to the levels of SAM. Such a system may acclimate cells for survival when SAM synthesis is reduced, whereas it may support proliferation when SAM is sufficient.     

Radical SAM enzymes involved in the biosynthesis of purine-based natural products

The radical S-adenosyl-l-methionine (SAM) superfamily is a widely distributed group of iron-sulfur containing proteins that exploit the reactivity of the high energy intermediate, 5'-deoxyadenosyl radical, which is produced by the reductive cleavage of SAM, to carry-out complex radical-mediated transformations. The reactions catalyzed by radical SAM enzymes range from simple group migrations to complex reactions in protein and RNA modification. This review will highlight three radical SAM enzymes that catalyze reactions involving modified guanosines in the biosynthesis pathways of the hypermodified tRNA base wybutosine; secondary metabolites of 7-deazapurine structure, including the hypermodified tRNA base queuosine; and the redox cofactor F(420). This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.     

Refolding of a fully functional flavivirus methyltransferase revealed that S‐adenosyl methionine but not S‐adenosyl homocysteine is copurified with flavivirus methyltransferase

Methylation of flavivirus RNA is vital for its stability and translation in the infected host cell. This methylation is mediated by the flavivirus methyltransferase (MTase), which methylates the N7 and 2′‐O positions of the viral RNA cap by using S‐adenosyl‐l ‐methionine (SAM) as a methyl donor. In this report, we demonstrate that SAM, in contrast to the reaction by‐product S‐adenosyl‐l ‐homocysteine, which was assumed previously, is copurified with the Dengue (DNV) and West Nile virus MTases produced in Escherichia coli (E. coli). This endogenous SAM can be removed by denaturation and refolding of the MTase protein. The refolded MTase of DNV serotype 3 (DNV3) displays methylation activity comparable to native enzyme, and its crystal structure at 2.1 Å is almost identical to that of native MTase. We characterized the binding of Sinefungin (SIN), a previously described SAM‐analog inhibitor of MTase function, to the native and refolded DNV3 MTase by isothermal titration calorimetry, and found that SIN binds to refolded MTase with more than 16 times the affinity of SIN binding to the MTase purified natively. Moreover, we show that SAM is also copurified with other flavivirus MTases, indicating that purification by refolding may be a generally applicable tool for studying flavivirus MTase inhibition.     

Evidence for a second class of S-adenosylmethionine riboswitches and other regulatory RNA motifs in alpha-proteobacteria

Background

Riboswitches are RNA elements in the 5' untranslated leaders of bacterial mRNAs that directly sense the levels of specific metabolites with a structurally conserved aptamer domain to regulate expression of downstream genes. Riboswitches are most common in the genomes of low GC Gram-positive bacteria (for example, Bacillus subtilis contains examples of all known riboswitches), and some riboswitch classes seem to be restricted to this group.

Results

We used comparative sequence analysis and structural probing to identify five RNA elements (serC, speF, suhB, ybhL, and metA) that reside in the intergenic regions of Agrobacterium tumefaciens and many other α-proteobacteria. One of these, the metA motif, is found upstream of methionine biosynthesis genes and binds S-adenosylmethionine (SAM). This natural aptamer most likely functions as a SAM riboswitch (SAM-II) with a consensus sequence and structure that is distinct from the class of SAM riboswitches (SAM-I) predominantly found in Gram-positive bacteria. The minimal functional SAM-II aptamer consists of fewer than 70 nucleotides, which form a single stem and a pseudoknot. Despite its simple architecture and lower affinity for SAM, the SAM-II aptamer strongly discriminates against related compounds.

Conclusion

SAM-II is the only metabolite-binding riboswitch class identified so far that is not found in Gram-positive bacteria, and its existence demonstrates that biological systems can use multiple RNA structures to sense a single chemical compound. The two SAM riboswitches might be 'RNA World' relics that were selectively retained in certain bacterial lineages or new motifs that have emerged since the divergence of the major bacterial groups.     

SAMMate: a GUI tool for processing short read alignments in SAM/BAM format

Background  

Next Generation Sequencing (NGS) technology generates tens of millions of short reads for each DNA/RNA sample. A key step in NGS data analysis is the short read alignment of the generated sequences to a reference genome. Although storing alignment information in the Sequence Alignment/Map (SAM) or Binary SAM (BAM) format is now standard, biomedical researchers still have difficulty accessing this information.     

Using S-adenosyl-l-homocysteine capture compounds to characterize S-adenosyl-l-methionine and S-adenosyl-l-homocysteine binding proteins

S-Adenosyl-l-methionine (SAM) is recognized as an important cofactor in a variety of biochemical reactions. As more proteins and pathways that require SAM are discovered, it is important to establish a method to quickly identify and characterize SAM binding proteins. The affinity of S-adenosyl-l-homocysteine (SAH) for SAM binding proteins was used to design two SAH-derived capture compounds (CCs). We demonstrate interactions of the proteins COMT and SAHH with SAH–CC with biotin used in conjunction with streptavidin–horseradish peroxidase. After demonstrating SAH-dependent photo-crosslinking of the CC to these proteins, we used a CC labeled with a fluorescein tag to measure binding affinity via fluorescence anisotropy. We then used this approach to show and characterize binding of SAM to the PR domain of PRDM2, a lysine methyltransferase with putative tumor suppressor activity. We calculated the K_d values for COMT, SAHH, and PRDM2 (24.1 ± 2.2 μM, 6.0 ± 2.9 μM, and 10.06 ± 2.87 μM, respectively) and found them to be close to previously established K_d values of other SAM binding proteins. Here, we present new methods to discover and characterize SAM and SAH binding proteins using fluorescent CCs.     

Structural basis for recognition of S-adenosylhomocysteine by riboswitches

S-adenosyl-(L)-homocysteine (SAH) riboswitches are regulatory elements found in bacterial mRNAs that up-regulate genes involved in the S-adenosyl-(L)-methionine (SAM) regeneration cycle. To understand the structural basis of SAH-dependent regulation by RNA, we have solved the structure of its metabolite-binding domain in complex with SAH. This structure reveals an unusual pseudoknot topology that creates a shallow groove on the surface of the RNA that binds SAH primarily through interactions with the adenine ring and methionine main chain atoms and discriminates against SAM through a steric mechanism. Chemical probing and calorimetric analysis indicate that the unliganded RNA can access bound-like conformations that are significantly stabilized by SAH to direct folding of the downstream regulatory switch. Strikingly, we find that metabolites bearing an adenine ring, including ATP, bind this aptamer with sufficiently high affinity such that normal intracellular concentrations of these compounds may influence regulation of the riboswitch.