MicroRNAs (miRNAs) constitute a large family of noncoding RNAs that function as guide molecules in diverse gene silencing pathways. Current efforts are focused on the regulatory function of miRNAs, while little is known about how these unusual genes themselves are regulated. Here we present the first direct evidence that miRNA genes are transcribed by RNA polymerase II (pol II). The primary miRNA transcripts (pri-miRNAs) contain cap structures as well as poly(A) tails, which are the unique properties of class II gene transcripts. The treatment of human cells with a-amanitin decreased the level of pri-miRNAs at a concentration that selectively inhibits pol II activity. Furthermore, chromatin immunoprecipitation analyses show that pol II is physically associated with a miRNA promoter. We also describe, for the first time, the detailed structure of a miRNA gene by determining the promoter and the terminator of mir-23aB27aB24-2. These data indicate that pol II is the main, if not the only, RNA polymerase for miRNA gene transcription. Our study offers a basis for understanding the structure and regulation of miRNA genes.

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We present a single molecule method for counting proteins within a diffraction-limited area when using photoactivated localization microscopy. The intrinsic blinking of photoactivatable fluorescent proteins mEos2 and Dendra2 leads to an overcounting error, which constitutes a major obstacle for their use as molecular counting tags. Here, we introduce a kinetic model to describe blinking and show that Dendra2 photobleaches three times faster and blinks seven times less than mEos2, making Dendra2 a better photoactivated localization microscopy tag than mEos2 for molecular counting. The simultaneous activation of multiple molecules is another source of error, but it leads to molecular undercounting instead. We propose a photoactivation scheme that maximally separates the activation of different molecules, thus helping to overcome undercounting. We also present a method that quantifies the total counting error and minimizes it by balancing over-and undercounting. This unique method establishes that Dendra2 is better for counting purposes than mEos2, allowing us to count in vitro up to 200 molecules in a diffraction-limited spot with a bias smaller than 2% and an uncertainty less than 6% within 10 min. Finally, we demonstrate that this counting method can be applied to protein quantification in vivo by counting the bacterial flagellar motor protein FliM fused to Dendra2.super-resolution optical microscopy | single molecule counting | fluorescence blinking K nowing the state of oligomerization of molecules can help to establish their structural organization in space and ultimately unravel their function. Electron microscopy, cryo-EM, and crystallography are the methods of choice to elucidate macromolecular structures at intermediate and high resolution, respectively, but these techniques often involve elaborate sample preparation or require isolating the protein complexes from their natural environment. Optical microscopy with fluorescent proteins provides a much less invasive alternative and allows quantification of proteins with single molecule sensitivity (1, 2). However, the resolution of conventional microscopy is diffraction-limited to approximately 250 nm, a dimension much larger than the size of protein complexes.In photoactivated localization microscopy (PALM), multiple fluorescent molecules spatially closer than the diffraction limit can be resolved by separating their contributions in time (3). PALM can, in principle, be used to count single molecules located within a diffraction-limited spot in the image. Ideally, any irreversibly photoactivatable fluorescent protein (PA-FP) can be used as a tag to count, for example, the number of proteins of a particular kind inside a cell, by simply tallying the number of emission bursts (4-6). In practice, however, the potential counting error incurred by the fluorescent blinking of PA-FPs in their photoactivated form (3, 6, 7) has been a major obstacle to the use of PALM for molecular counting. Although a semiempirical approach to correct the artifact due to the mEos2...

The molecular mechanisms underlying angioimmunoblastic T cell lymphoma (AITL), a common type of mature T cell lymphoma of poor prognosis, are largely unknown. Here we report a frequent somatic mutation in RHOA (encoding p.Gly17Val) using exome and transcriptome sequencing of samples from individuals with AITL. Further examination of the RHOA mutation encoding p.Gly17Val in 239 lymphoma samples showed that the mutation was specific to T cell lymphoma and was absent from B cell lymphoma. We demonstrate that the RHOA mutation encoding p.Gly17Val, which was found in 53.3% (24 of 45) of the AITL cases examined, is oncogenic in nature using multiple molecular assays. Molecular modeling and docking simulations provided a structural basis for the loss of GTPase activity in the RHOA Gly17Val mutant. Our experimental data and modeling results suggest that the RHOA mutation encoding p.Gly17Val is a driver mutation in AITL. On the basis of these data and through integrated pathway analysis, we build a comprehensive signaling network for AITL oncogenesis.