Michael J. Moore

Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus

Spike (S) proteins of coronaviruses, including the coronavirus that causes severe acute respiratory syndrome (SARS), associate with cellular receptors to mediate infection of their target cells. Here we identify a metallopeptidase, angiotensin-converting enzyme 2 (ACE2), isolated from SARS coronavirus (SARS-CoV)-permissive Vero E6 cells, that efficiently binds the S1 domain of the SARS-CoV S protein. We found that a soluble form of ACE2, but not of the related enzyme ACE1, blocked association of the S1 domain with Vero E6 cells. 293T cells transfected with ACE2, but not those transfected with human immunodeficiency virus-1 receptors, formed multinucleated syncytia with cells expressing S protein. Furthermore, SARS-CoV replicated efficiently on ACE2-transfected but not mock-transfected 293T cells. Finally, anti-ACE2 but not anti-ACE1 antibody blocked viral replication on Vero E6 cells. Together our data indicate that ACE2 is a functional receptor for SARS-CoV.

Receptor and viral determinants of SARS-coronavirus adaptation to human ACE2

Human angiotensin‐converting enzyme 2 (ACE2) is a functional receptor for SARS coronavirus (SARS‐CoV). Here we identify the SARS‐CoV spike (S)‐protein‐binding site on ACE2. We also compare S proteins of SARS‐CoV isolated during the 2002–2003 SARS outbreak and during the much less severe 2003–2004 outbreak, and from palm civets, a possible source of SARS‐CoV found in humans. All three S proteins bound to and utilized palm‐civet ACE2 efficiently, but the latter two S proteins utilized human ACE2 markedly less efficiently than did the S protein obtained during the earlier human outbreak. The lower affinity of these S proteins could be complemented by altering specific residues within the S‐protein‐binding site of human ACE2 to those of civet ACE2, or by altering S‐protein residues 479 and 487 to residues conserved during the 2002–2003 outbreak. Collectively, these data describe molecular interactions important to the adaptation of SARS‐CoV to human cells, and provide insight into the severity of the 2002–2003 SARS epidemic.

An Alternative Splicing Network Links Cell-Cycle Control to Apoptosis

Alternative splicing is a vast source of biological regulation and diversity that is misregulated in cancer and other diseases. To investigate global control of alternative splicing in human cells, we analyzed splicing of mRNAs encoding Bcl2 family apoptosis factors in a genome-wide siRNA screen. The screen identified many regulators of Bcl-x and Mcl1 splicing, notably an extensive network of cell-cycle factors linked to aurora kinase A. Drugs or siRNAs that induce mitotic arrest promote proapoptotic splicing of Bcl-x, Mcl1, and caspase-9 and alter splicing of other apoptotic transcripts. This response precedes mitotic arrest, indicating coordinated upregulation of prodeath splice variants that promotes apoptosis in arrested cells. These shifts correspond to posttranslational turnover of splicing regulator ASF/SF2, which directly binds and regulates these target mRNAs and globally regulates apoptosis. Broadly, our results reveal an alternative splicing network linking cell-cycle control to apoptosis.

Sulphated tyrosines mediate association of chemokines and Plasmodium vivax Duffy binding protein with the Duffy antigen/receptor for chemokines (DARC).

Plasmodium vivax is one of four Plasmodium species that cause human malaria. P. vivax and a related simian malaria parasite, Plasmodium knowlesi, invade erythrocytes by binding the Duffy antigen/receptor for chemokines (DARC) through their respective Duffy binding proteins. Here we show that tyrosines 30 and 41 of DARC are modified by addition of sulphate groups, and that the sulphated tyrosine 41 is essential for association of the Duffy binding proteins of P. vivax (PvDBP) and P. knowlesi (PkDaBP) with DARC‐expressing cells. These sulphated tyrosines also participate in the association of DARC with each of its four known chemokine ligands. Alteration of tyrosine 41 to phenylalanine interferes with MCP‐1, RANTES and MGSA association with DARC, but not with that of IL8. In contrast, alteration of tyrosine 30 to phenylalanine interferes with the association of IL8 with DARC. A soluble sulphated amino‐terminal domain of DARC, but not one modified to phenylalanine at residue 41, can be used to block the association of PvDBP and PkDaBP with red blood cells, with an IC50 of approximately 5 nM. These data are consistent with a role for tyrosine sulphation in the association of many or most chemokines with their receptors, and identify a key molecular determinant of erythrocyte invasion by P. vivax.

Mapping Argonaute and conventional RNA-binding protein interactions with RNA at single-nucleotide resolution using HITS-CLIP and CIMS analysis

The identification of sites where RNA-binding proteins (RNABPs) interact with target RNAs opens the door to understanding the vast complexity of RNA regulation. UV cross-linking and immunoprecipitation (CLIP) is a transformative technology in which RNAs purified from in vivo cross-linked RNA-protein complexes are sequenced to reveal footprints of RNABP:RNA contacts. CLIP combined with high-throughput sequencing (HITS-CLIP) is a generalizable strategy to produce transcriptome-wide maps of RNA binding with higher accuracy and resolution than standard RNA immunoprecipitation (RIP) profiling or purely computational approaches. The application of CLIP to Argonaute proteins has expanded the utility of this approach to mapping binding sites for microRNAs and other small regulatory RNAs. Finally, recent advances in data analysis take advantage of cross-link–induced mutation sites (CIMS) to refine RNA-binding maps to single-nucleotide resolution. Once IP conditions are established, HITS-CLIP takes ∼8 d to prepare RNA for sequencing. Established pipelines for data analysis, including those for CIMS, take 3–4 d.

miRNA–target chimeras reveal miRNA 3′-end pairing as a major determinant of Argonaute target specificity

microRNAs (miRNAs) act as sequence-specific guides for Argonaute (AGO) proteins, which mediate posttranscriptional silencing of target messenger RNAs. Despite their importance in many biological processes, rules governing AGO–miRNA targeting are only partially understood. Here we report a modified AGO HITS-CLIP strategy termed CLEAR (covalent ligation of endogenous Argonaute-bound RNAs)-CLIP, which enriches miRNAs ligated to their endogenous mRNA targets. CLEAR-CLIP mapped ∼130,000 endogenous miRNA–target interactions in mouse brain and ∼40,000 in human hepatoma cells. Motif and structural analysis define expanded pairing rules for over 200 mammalian miRNAs. Most interactions combine seed-based pairing with distinct, miRNA-specific patterns of auxiliary pairing. At some regulatory sites, this specificity confers distinct silencing functions to miRNA family members with shared seed sequences but divergent 3′-ends. This work provides a means for explicit biochemical identification of miRNA sites in vivo, leading to the discovery that miRNA 3′-end pairing is a general determinant of AGO binding specificity.

PQBP1, a factor linked to intellectual disability, affects alternative splicing associated with neurite outgrowth

Polyglutamine-binding protein 1 (PQBP1) is a highly conserved protein associated with neurodegenerative disorders. Here, we identify PQBP1 as an alternative messenger RNA (mRNA) splicing (AS) effector capable of influencing splicing of multiple mRNA targets. PQBP1 is associated with many splicing factors, including the key U2 small nuclear ribonucleoprotein (snRNP) component SF3B1 (subunit 1 of the splicing factor 3B [SF3B] protein complex). Loss of functional PQBP1 reduced SF3B1 substrate mRNA association and led to significant changes in AS patterns. Depletion of PQBP1 in primary mouse neurons reduced dendritic outgrowth and altered AS of mRNAs enriched for functions in neuron projection development. Disease-linked PQBP1 mutants were deficient in splicing factor associations and could not complement neurite outgrowth defects. Our results indicate that PQBP1 can affect the AS of multiple mRNAs and indicate specific affected targets whose splice site determination may contribute to the disease phenotype in PQBP1-linked neurological disorders.

Localization of theta alloantigens in mouse brain by immunofluorescence and cytotoxic inhibition

Summary The indirect fluorescent antibody and the cytotoxic inhibition techniques were used to localize theta alloantigens in adult mouse brain. The procedure was validated critically by genetic specification of brain tissue and antiserum, as well as by the standard immunologic blocking and deletion tests. Reactive sites were demonstrated in white matter tracts such as corpus callosum, internal capsule, penetrating tracts of the caudate/putamen, fimbria of the hippocampal formation and central white matter at the cerebellar folia. In addition reactive sites were demonstrated in cells of various sizes from the cerebral gray matter. Both the white and gray matter localizations were confirmed by cytotoxic inhibition tests.

PET/CT in Abdominal and Pelvic Malignancies: Principles and Practices

Current multi-detector computed tomography (MDCT) allows for rapid acquisition of data sets with accurate anatomic detail and high spatial resolution. This provides valuable multi-planar information regarding the morphologic features and attenuation values of both normal anatomic structures and pathologic lesions. Since its introduction more than 25 years ago, CT has become widely used and can be considered the modality of choice for much cross-sectional imaging, particularly of oncologic entities. A limitation of CT, however, is its reliance on morphologic changes in the size, shape, or attenuation values of a structure to detect pathologic processes. Therefore, CT is less sensitive in the imaging of early disease processes and in the detection of disease recurrence in tissues that are already morphologically abnormal as a result of treatment.

Global mapping of miRNA-target interactions in cattle ( Bos taurus )

With roles in development, cell proliferation and disease, micro-RNA (miRNA) biology is of great importance and a potential therapeutic target. Here we used cross-linking immunoprecipitation (CLIP) and ligation of miRNA-target chimeras on the Argonaute (AGO) protein to globally map miRNA interactions in the cow. The interactome is the deepest reported to date. miRNA targeting principles are consistent with observations in other species, but with expanded pairing rules. Experimental mapping robustly predicted functional miR-17 regulatory sites. From miRNA-specific targeting for >5000 mRNAs we determined gene ontologies (GO). This confirmed repression of genes important for embryonic development and cell cycle progress by the let-7 family, and repression of those involved in cell cycle arrest by the miR-17 family, but also suggested a number of unappreciated miRNA functions. Our results provide a significant resource for understanding of bovine and species-conserved miRNA regulation, and demonstrate the power of experimental methods for establishing comprehensive interaction maps.