HEN1 recognizes 21-24 nt small RNA duplexes and deposits a methyl group onto the 2' OH of the 3' terminal nucleotide.

microRNAs (miRNAs) and small interfering RNAs (siRNAs) in plants bear a methyl group on the ribose of the 3' terminal nucleotide. We showed previously that the methylation of miRNAs and siRNAs requires the protein HEN1 in vivo and that purified HEN1 protein methylates miRNA/miRNA* duplexes in vitro. In this study, we show that HEN1 methylates both miRNA/miRNA* and siRNA/siRNA* duplexes in vitro with a preference for 21-24 nt RNA duplexes with 2 nt overhangs. We also demonstrate that HEN1 deposits the methyl group on to the 2' OH of the 3' terminal nucleotide. Among various modifications that can occur on the ribose of the terminal nucleotide, such as 2'-deoxy, 3'-deoxy, 2'-O-methyl and 3'-O-methyl, only 2'-O-methyl on a small RNA inhibits the activity of yeast poly(A) polymerase (PAP). These findings indicate that HEN1 specifically methylates miRNAs and siRNAs and implicate the importance of the 2'-O-methyl group in the biology of RNA silencing

Nonradioactive, ultrasensitive site-specific protein–protein photocrosslinking: interactions of α-helix 2 of TATA-binding protein with general transcription factor TFIIA and transcriptional repressor NC2

We have developed an approach that enables nonradioactive, ultrasensitive (attamole sensitivity) site-specific protein–protein photocrosslinking, and we have applied the approach to the analysis of interactions of α-helix 2 (H2) of human TATA-element binding protein (TBP) with general transcription factor TFIIA and transcriptional repressor NC2. We have found that TBP H2 can be crosslinked to TFIIA in the TFIIA–TBP–DNA complex and in higher order transcription–initiation complexes, and we have mapped the crosslink to the ‘connector’ region of the TFIIA α/β subunit (TFIIAα/β). We further have found that TBP H2 can be crosslinked to NC2 in the NC2–TBP–DNA complex, and we have mapped the crosslink to the C-terminal ‘tail’ of the NC2 α-subunit (NC2α). Interactions of TBP H2 with the TFIIAα/β connector and the NC2α C-terminal tail were not observed in crystal structures of TFIIA–TBP–DNA and NC2–TBP–DNA complexes, since relevant segments of TFIIA and NC2 were not present in truncated TFIIA and NC2 derivatives used for crystallization. We propose that interactions of TBP H2 with the TFIIAα/β connector and the NC2α C-terminal tail provide an explanation for genetic results suggesting importance of TBP H2 in TBP–TFIIA interactions and TBP–NC2 interactions, and provide an explanation—steric exclusion—for competition between TFIIA and NC2

HEN1 recognizes 21–24 nt small RNA duplexes and deposits a methyl group onto the 2\u27 OH of the 3\u27 terminal nucleotide

microRNAs (miRNAs) and small interfering RNAs (siRNAs) in plants bear a methyl group on the ribose of the 3\u27 terminal nucleotide. We showed previously that the methylation of miRNAs and siRNAs requires the protein HEN1 in vivo and that purified HEN1 protein methylates miRNA/miRNA* duplexes in vitro. In this study, we show that HEN1 methylates both miRNA/miRNA* and siRNA/siRNA* duplexes in vitro with a preference for 21–24 nt RNA duplexes with 2 nt overhangs. We also demonstrate that HEN1 deposits the methyl group on to the 2\u27 OH of the 3\u27 terminal nucleotide. Among various modifications thatcanoccuronthe ribose of the terminal nucleotide, such as 2\u27-deoxy, 3\u27-deoxy, 2\u27-O-methyl and 3\u27-O-methyl, only 2\u27-O-methyl on a small RNA inhibits the activity of yeast poly(A) polymerase (PAP). These findings indicate that HEN1 specificallymethylates miRNAs and siRNAs and implicate the importance of the 2\u27-O-methyl group in the biology of RNA silencing

HEN1 recognizes 21-24 nt small RNA duplexes and deposits a methyl group onto the 2' OH of the 3' terminal nucleotide.

microRNAs (miRNAs) and small interfering RNAs (siRNAs) in plants bear a methyl group on the ribose of the 3' terminal nucleotide. We showed previously that the methylation of miRNAs and siRNAs requires the protein HEN1 in vivo and that purified HEN1 protein methylates miRNA/miRNA* duplexes in vitro. In this study, we show that HEN1 methylates both miRNA/miRNA* and siRNA/siRNA* duplexes in vitro with a preference for 21-24 nt RNA duplexes with 2 nt overhangs. We also demonstrate that HEN1 deposits the methyl group on to the 2' OH of the 3' terminal nucleotide. Among various modifications that can occur on the ribose of the terminal nucleotide, such as 2'-deoxy, 3'-deoxy, 2'-O-methyl and 3'-O-methyl, only 2'-O-methyl on a small RNA inhibits the activity of yeast poly(A) polymerase (PAP). These findings indicate that HEN1 specifically methylates miRNAs and siRNAs and implicate the importance of the 2'-O-methyl group in the biology of RNA silencing

Methyltransferase reaction by GST-HEN1 on siRNA/siRNA* duplexes and miRNA/miRNA* duplexes with 1–5 3′ overhangs

<p><b>Copyright information:</b></p><p>Taken from "HEN1 recognizes 21–24 nt small RNA duplexes and deposits a methyl group onto the 2′ OH of the 3′ terminal nucleotide"</p><p>Nucleic Acids Research 2006;34(2):667-675.</p><p>Published online 30 Jan 2006</p><p>PMCID:PMC1356533.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> All duplexes in this figure were methylated with the same GST-HEN1 preparation and are therefore comparable to one another. The numbers above the lanes correspond to those in

Effect of various modifications on the 3′ terminal nucleotide of a small RNA on T4 RNA ligase- and yeast PAP-catalyzed reactions

<p><b>Copyright information:</b></p><p>Taken from "HEN1 recognizes 21–24 nt small RNA duplexes and deposits a methyl group onto the 2′ OH of the 3′ terminal nucleotide"</p><p>Nucleic Acids Research 2006;34(2):667-675.</p><p>Published online 30 Jan 2006</p><p>PMCID:PMC1356533.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> () T4 RNA ligase-mediated ligation of various miR173 forms to an RNA linker. () Activity of yeast PAP on various forms of miR173 in the presence of 2 pmol [α-P]-ATP. The ladders or smears represent products of PAP-catalyzed reaction. () Activity of yeast PAP on various forms of miR173 in the presence of 10 pmol [α-P]-ATP

Transcription inhibition by the depsipeptide antibiotic salinamide A.

We report that bacterial RNA polymerase (RNAP) is the functional cellular target of the depsipeptide antibiotic salinamide A (Sal), and we report that Sal inhibits RNAP through a novel binding site and mechanism. We show that Sal inhibits RNA synthesis in cells and that mutations that confer Sal-resistance map to RNAP genes. We show that Sal interacts with the RNAP active-center 'bridge-helix cap' comprising the 'bridge-helix N-terminal hinge', 'F-loop', and 'link region'. We show that Sal inhibits nucleotide addition in transcription initiation and elongation. We present a crystal structure that defines interactions between Sal and RNAP and effects of Sal on RNAP conformation. We propose that Sal functions by binding to the RNAP bridge-helix cap and preventing conformational changes of the bridge-helix N-terminal hinge necessary for nucleotide addition. The results provide a target for antibacterial drug discovery and a reagent to probe conformation and function of the bridge-helix N-terminal hinge.DOI: http://dx.doi.org/10.7554/eLife.02451.001