MicroRNAs (miRNAs) regulate the expression of a large number of protein-coding genes. Their primary transcripts (pri-miRNAs) have to undergo multiple processing steps to reach the functional form. Little is known about how the processing of miRNAs is modulated. Here we show that the RNA-binding protein DiGeorge critical region-8 (DGCR8), which is essential for the first processing step, is a heme-binding protein. The association with heme promotes dimerization of DGCR8. The heme-bound DGCR8 dimer seems to trimerize upon binding pri-miRNAs and is active in triggering pri-miRNA cleavage, whereas the heme-free monomer is much less active. A heme-binding region of DGCR8 inhibits the pri-miRNA-processing activity of the monomer. This putative autoinhibition is overcome by heme. Our finding that heme is involved in pri-miRNA processing suggests that the gene-regulation network of miRNAs and signal-transduction pathways involving heme might be connected.

The addition of m-nitrobenzyl alcohol (m-NBA) was shown previously (Lomeli et al., J. Am. Soc. Mass Spectrom. 2009, 20, 593-596) to enhance multiple charging of native proteins and noncovalent protein complexes in electrospray ionization (ESI) mass spectra. Additional new reagents have been found to "supercharge" proteins from nondenaturing solutions; several of these reagents are shown to be more effective than m-NBA for increasing positive charging. Using the myoglobin protein-protoporphyrin IX (heme) complex, the following reagents were shown to increase ESI charging : benzyl alcohol, m-nitroacetophenone, m-nitrobenzonitrile, o-NBA, m-NBA, p-NBA, m-nitrophenyl ethanol, sulfolane (tetramethylene sulfone), and m-(trifluoromethyl)-benzyl alcohol. Based on average charge state, sulfolane displayed a greater charge increase (61%) than m-NBA (21%) for myoglobin in aqueous solutions. The reagents that promote higher ESI charging appear to have low solution-phase basicities and relatively low gas-phase basicities, and are less volatile than water. Another feature of mass spectra from some of the active reagents is that adducts are present on higher charge states, suggesting that a mechanism by which proteins acquire additional charge involves direct interaction with the reagent, in addition to other factors such as surface tension and protein denaturation. (J Am Soc Mass Spectrom 2010, 21, 127-131) © 2010 American Society for Mass Spectrometry E lectrospray ionization is distinguished among other desorption/ionization sources for mass spectrometry by its ready generation of multiply charged molecules, the value of which was recognized early by Fenn's group: "This feature is very attractive in that it extends the effective mass range of any analyzer by a factor equal to the number of charges per ion. Moreover, because the ions have lower m/z values, they are generally easier to detect and weigh than are singly charged ions of the same mass [1]."After the utility of ESI-MS for protein analysis was demonstrated, multiple charging was rapidly extended to tandem mass spectrometry of large peptides and proteins [2,3]. Dissociation efficiency of large molecules is enhanced with increasing charge, allowing sequenceinformative product ions to be measured. Early ESI-MS studies investigated the potential of different solvents to increase charging [4]. However, useful enhancement of ESI multiple charging was not described clearly until Iavarone and Williams reported "supercharging" promoted by agents such as m-nitrobenzyl alcohol (m-NBA) [5,6]. For protein analyses, their work largely investigated solutions considered to be denaturing.Recently, we demonstrated that multiple charging of noncovalent protein complexes could be increased in ESI-MS when spectra are obtained from nondenaturing protein solutions containing up to 1% (vol/vol) m-NBA [7]. Increases in charge ranged from 8% for the 690 kDa 20S proteasome complex to 48% additional charge for zinc-bound 29 kDa carbonic anhydrase protein. These protein structures were cons...

IntroductionMaintenance of the hematopoietic system requires continual replenishment of mature blood cells from a rare population of bone marrow residing hematopoietic stem cells (HSCs). The alteration of the homeostatic control of hematopoiesis is considered to be a major culprit of drastic increase in pathologic incidences, such as bone marrow failure, anemia, and myeloid leukemia during aging. 1 However, the underlying mechanisms of pathogenesis of hematologic malignancy in elderly population remain poorly understood.Mounting evidence supports the idea that the accumulation of somatic DNA damage is a main cause of aging in multicellular organisms. [2][3][4][5] Mice with mutations in various DNA repair genes exhibit accelerated aging in the hematopoietic system because of the premature exhaustion of HSCs, indicating that DNA repair is crucial for the maintenance of HSC self-renewal and hematopoietic function. 6,7 DNA damage can directly result from genotoxic treatment such as ionizing radiation (IR), or may simply occur as a consequence of genome duplication infidelity or of genotoxic effects of reactive oxygen species (ROS). ROS, such as superoxide anions and hydrogen peroxide, are byproducts of normal oxidative metabolism in eukaryotic cells and are involved in many signaling process. However, they can be harmful to cellular components, including DNA. 3,8,9 An uncontrolled elevation of intracellular ROS levels is believed to contribute to cellular aging and the senescence process. 3 In fact, an abnormal elevation of intracellular ROS levels has been implicated in the pathogenesis of various diseases, such as ataxia telangiectasia and Fanconi anemia. 3 In that sense, the maintenance of ROS levels, through highly regulated mechanisms, is essential for cellular homeostasis. 10 Being continuously exposed to oxidants produced during metabolic activity and to external oxidants or oxidant-inducers through normal cellular physiology, DNAs within cells inevitably suffer the oxidative damage. Therefore, an accelerated proliferation of hematopoietic cells, which is expected to occur after clinical HSC transplantation, might lead to DNA damage through overexposure to oxidative stress generated on each cell cycle. Indeed, a hyperproliferation caused an accumulation of oxidative stress and resulted in functional exhaustion of murine HSCs, as shown by the failure to reconstitute hematopoiesis after serial transplantations. 11 Taken together, we hypothesize that the continuous production of ROS during long-term repopulation induces an accumulation of genomic damage that leads to exhaustion of human HSCs. We have previously developed a strategy that enables to examine the multipotency of a single human HSC using a reliable surrogate system. 12,13 By determining the in vivo repopulating dynamics of individual human HSCs, we demonstrated that the repopulating potential of the majority of human HSCs progressively deteriorated as they underwent extensive repopulation process. Furthermore, the self-renewing long-term repopulating...

Mass spectrometry (MS) with electrospray ionization (ESI) has the capability to measure and detect noncovalent protein-ligand and protein-protein complexes. However, information on the sites of ligand binding is not easily obtained by the ESI-MS methodology. Electron capture dissociation (ECD) favors cleavage of covalent backbone bonds of protein molecules. We show that this characteristic of ECD translates to noncovalent protein-ligand complexes, as covalent backbone bonds of protein complexes are dissociated, but the noncovalent ligand interaction is retained. For the complex formed from 140-residue, 14.5 kDa alpha-synuclein protein, and one molecule of polycationic spermine (202 Da), ECD generates product ions that retain the protein-spermine noncovalent interaction. Spermine binding is localized to residues 106-138; the ECD data are consistent with previous solution NMR studies. Our studies suggest that ECD mass spectrometry can be used to determine directly the sites of ligand binding to protein targets.

Increased multiple charging of native proteins and noncovalent protein complexes is observed in electrospray ionization (ESI) mass spectra obtained from nondenaturing protein solutions containing up to 1% (v/v) m-nitrobenzyl alcohol (m-NBA). The increases in charge ranged from 8% for the 690 kDa α 7 β 7 β 7 α 7 20S proteasome complex to 48% additional charge for the zinc-bound 29 kDa carbonic anhydrase-II protein. No dissociation of the noncovalently bound ligands/subunits was observed upon the addition of m-NBA. It is not clear if the enhanced charging is related to altered surface tension as proposed in the "supercharging" model of Iavarone and Williams (Iavarone, A. T.; Williams, E. R. J. Am. Chem. Soc. 2003, 125, 2319-2327. However, more highly charged noncovalent protein complexes have utility in relaxing slightly the mass-to-charge (m/z) requirements of the mass spectrometer for detection and will be effective for enhancing the efficiency for tandem mass spectrometry studies of protein complexes.