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The 5' N

A hallmark of eukaryotic mRNAs has long been the 5'-end m7G cap. This paradigm was recently amended by recent reports that Saccharomyces cerevisiae and mammalian cells also contain mRNAs carrying a novel nicotinamide adenine dinucleotide (NAD+) cap at their 5'-end. The presence of an NAD+ cap on mRNA uncovers a previously unknown mechanism for controlling gene expression through nucleotide metabolite-directed mRNA turnover. In contrast to the m7G cap that stabilizes mRNA, the NAD+ cap targets RNA for rapid decay in mammalian cells through the DXO non-canonical decapping enzyme which removes intact NAD+ from RNA in a process termed 'deNADding'. This review highlights the identification of NAD+ caps, their mode of addition, and their functional significance in cells.

Recently, we reported that two homologous yeast proteins, Rai1 and Dxo1, function in a quality control mechanism to clear cells of incompletely 5' end-capped messenger RNAs (mRNAs). Here, we report that their mammalian homolog, Dom3Z (referred to as DXO), possesses pyrophosphohydrolase, decapping, and 5'-to-3' exoribonuclease activities. Surprisingly, we found that DXO preferentially degrades defectively capped pre-mRNAs in cells. Additional studies show that incompletely capped pre-mRNAs are inefficiently spliced at all introns, a fact that contrasts with current understanding, and are also poorly cleaved for polyadenylation. Crystal structures of DXO in complex with substrate mimic and products at a resolution of up to 1.5Å provide elegant insights into the catalytic mechanism and molecular basis for their three apparently distinct activities. Our data reveal a pre-mRNA 5' end capping quality control mechanism in mammalian cells, indicating DXO as the central player for this mechanism, and demonstrate an unexpected intimate link between proper 5' end capping and subsequent pre-mRNA processing.

Two major decapping enzymes are involved in the decay of eukaryotic mRNA, Dcp2 and DcpS. Despite the detection of robust DcpS decapping activity in cell extract, minimal to no decapping is detected from human Dcp2 (hDcp2) in extract. We now demonstrate that one reason for the lack of detectable hDcp2 activity in extract is due to the presence of inhibitory trans factor(s). Furthermore, we demonstrate that a previously identified testis-specific protein of unknown function implicated in nonspecific X-linked mental retardation, VCX-A, can function as an inhibitor of hDcp2 decapping in vitro and in cells. VCX-A is a noncanonical cap-binding protein that binds to capped RNA but not cap structure lacking an RNA. Its cap association is enhanced by hDcp2 to further augment the ability of VCX-A to inhibit decapping. Our data demonstrate that VCX-A can regulate mRNA stability and that it is an example of a tissue-specific decapping regulator.

Many methods are available and widely used to determine specific proteins that bind to a particular RNA of interest. However, approaches to identify unknown substrate RNAs to which an RNA-binding protein binds and potentially regulates are not as common. In this article we describe a technique termed isolation of specific nucleic acids associated with proteins (SNAAP) that allows the identification of mRNAs associated with a protein. Methods are detailed for expressing and purifying fusion proteins that are used to isolate substrate mRNPs employing differential display technology. Lastly, experiments are described to confirm that the RNAs identified are indeed bonafide substrates for an RNA-binding protein. As the number of known RNA-binding proteins increases, of which many are involved in genetic disorders, it is essential that methodologies exist to identify RNA-protein interactions to better understand the manifestation of disease.

Mechanistic understanding of mammalian mRNA turnover remains incomplete. We demonstrate that the 3′ to 5′ exoribonuclease decay pathway is a major contributor to mRNA decay both in cells and in cell extract. An exoribonuclease-dependent scavenger decapping activity was identified that follows decay of the mRNA and hydrolyzes the residual cap. The decapping activity is associated with a subset of the exosome proteins in vivo, implying a higher-order degradation complex consisting of exoribonucleases and a decapping activity, which together coordinate the decay of an mRNA. These findings indicate that following deadenylation of mammal mRNA, degradation proceeds by a coupled 3′ to 5′ exoribonucleolytic activity and subsequent hydrolysis of the cap structure by a scavenger decapping activity.

Regulation of mRNA turnover is a critical control mechanism of gene expression and is influenced by ribonucleoprotein (RNP) complexes that form on cis elements. All mRNAs have an intrinsic half-life and in many cases these half-lives can be altered by a variety of stimuli that are manifested through the formation or disruption of an RNP structure. The stability of alpha-globin mRNA is determined by elements in the 3' untranslated region that are bound by an RNP complex (alpha-complex) which appears to control the erythroid-specific accumulation of alpha-globin mRNA. The alpha-complex could consist of up to six distinct proteins or protein families. One of these families is a prominent polycytidylate binding activity which consists of two highly homologous proteins, alpha-complex proteins 1 and 2 (alphaCP1 and alphaCP2). This article focuses on various methodologies for the detection and manipulation of alphaCP1 and alphaCP2 binding to RNA and details means of isolating and characterizing mRNA bound by these proteins to study mRNA turnover and its regulation.

The tunneling of holes in a double-barrier GaAs/GaAlAs heterostructure, and interband tunneling in an InAs/AlSb/GaSb double-barrier heterostructure were calculated. A ten-band tight-binding model was used in order to incorporate mixing between the bands. The model was solved using a new method which allows many-layer structures to be handled without numerical instabilities. We have found that mixing between bands has a significant effect on the current-voltage characteristics, especially with the inclusion of the dependence of the transmission on the parallel component of the incident wavevecor ( k ∥ ). The current peaks can change by more than an order of magnitude, the heavy and light hole peaks can switch in magnitude, and peaks which are forbidden without mixing can appear.

Osteoblasts express high levels of liver/bone/kidney alkaline phosphatase (LBK AP), an enzyme critical for bone formation. Other tissues and cell types generally express much lower levels of LBK AP and correspondingly lower levels of mRNA. In light of our early observations that the human LBK AP promoter is expressed equally when transfected into a variety of different cells, we have carried out a detailed study of LBK AP gene expression in Saos-2 cells which are osteoblast-derived and express high levels of LBK AP mRNA, and in HepG2 hepatoblastoma cells which express LBK AP mRNA at levels which are approximately 1000-fold lower. Our results indicate that both of these cells utilize the same promoter sequences to initiate transcription of their LBK AP genes at roughly the same rates. Moreover, the stability of cytoplasmic LBK AP mRNA is equal in both cell types. The lack of any apparent buildup of unspliced precursor mRNA in the nucleus of HepG2 cells leads us to the conclusion that splicing (and nuclear export) is equivalent. It is therefore likely that differential expression is controlled at a very early step post-transcription, possibly by sequences that destabilize the nascent RNA in HepG2 cells. We reason that these destabilizing sequences are located in the gene's introns because a transfected LBK AP minigene, comprised of the full length cDNA and flanking sequences, is expressed efficiently in both cell types.

In this issue of Structure, Scarsdale et al. (2006) report structures of the Xenopus X29 Nudix decapping protein, including homodimer structures in complex with cap nucleotides. These structures reveal insights into the mechanism of cap substrate recognition and predict an RNA binding path on the protein surface.