Biomedical subjects

Xinfu Jiao

Publications and source records attributed to Xinfu Jiao.

8 recordsLinked to original sources

PubMed2006

Identification of an mRNA-decapping regulator implicated in X-linked mental retardation.

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.

Cell Line↗

PubMed2006

Identification of mRNA bound to RNA binding proteins by differential display.

A large number of RNA binding proteins have recently been identified that influence various human genetic disorders. However, the specific function of many of these proteins and what role they may play in a particular disease remains unclear. Identification of the substrate mRNA bound by an RNA binding protein will provide insights into the function of that protein and how its aberrant expression could lead to a disease phenotype. We have developed a technique termed SNAAP, for isolation of specific nucleic acids associated with proteins, to identify natural mRNA substrates for an RNA binding protein. The technique couples affinity purification of specific mRNAs bound by an RNA binding protein, with the identification of that mRNA using differential display (DD). Methods are described herein for the isolation and identification of endogenous mRNAs bound by any RNA binding protein, as well as methodology to validate the specificity of the binding. The availability of technologies to isolate the cognate substrate mRNAs potentially bound and regulated by an RNA binding protein involved in genetic disorders will greatly expedite our etiological understanding of the disorder and provide modalities for intervention.

3' Untranslated Regions↗

PubMed2005

Dcp2 Decaps m2,2,7GpppN-capped RNAs, and its activity is sequence and context dependent.

Hydrolysis of the mRNA cap plays a pivotal role in initiating and completing mRNA turnover. In nematodes, mRNA metabolism and cap-interacting proteins must deal with two populations of mRNAs, spliced leader trans-spliced mRNAs with a trimethylguanosine cap and non-trans-spliced mRNAs with a monomethylguanosine cap. We describe here the characterization of nematode Dcp1 and Dcp2 proteins. Dcp1 was inactive in vitro on both free cap and capped RNA and did not significantly enhance Dcp2 activity. Nematode Dcp2 is an RNA-decapping protein that does not bind cap and is not inhibited by cap analogs but is effectively inhibited by competing RNA irrespective of RNA sequence and cap. Nematode Dcp2 activity is influenced by both 5' end sequence and its context. The trans-spliced leader sequence on mRNAs reduces Dcp2 activity approximately 10-fold, suggesting that 5'-to-3' turnover of trans-spliced RNAs may be regulated. Nematode Dcp2 decaps both m(7)GpppG- and m(2,2,7)GpppG-capped RNAs. Surprisingly, both budding yeast and human Dcp2 are also active on m(2,2,7)GpppG-capped RNAs. Overall, the data suggest that Dcp2 activity can be influenced by both sequence and context and that Dcp2 may contribute to gene regulation in multiple RNA pathways, including monomethyl- and trimethylguanosine-capped RNAs.

Animals↗

PubMed2004

Functional analysis of mRNA scavenger decapping enzymes.

Eukaryotic cells primarily utilize exoribonucleases and decapping enzymes to degrade their mRNA. Two major decapping enzymes have been identified. The hDcp2 protein catalyzes hydrolysis of the 5' cap linked to an RNA moiety, whereas the scavenger decapping enzyme, DcpS, functions on a cap structure lacking the RNA moiety. DcpS is a member of the histidine triad (HIT) family of hydrolases and catalyzes the cleavage of m7GpppN. HIT proteins are homodimeric and contain two conserved 100-amino-acid HIT fold domains with independent active sites that are each sufficient to bind and hydrolyze cognate substrates. We carried out a functional characterization of the DcpS enzyme and demonstrate that unlike previously described HIT proteins, DcpS is a modular protein that requires both the core HIT fold at the carboxyl-terminus and sequences at the amino-terminus of the protein for cap binding and hydrolysis. Interestingly, DcpS can efficiently compete for and hydrolyze the cap structure even in the presence of excess eIF4E, implying that DcpS could function to alleviate the accumulation of complexes between eIF4E and cap structure that would otherwise accumulate following mRNA decay. Using immunofluorescence microscopy, we demonstrate that DcpS is predominantly a nuclear protein, with low levels of detected protein in the cytoplasm. Furthermore, analysis of the endogenous hDcp2 protein reveals that in addition to the cytoplasmic foci, it is also present in the nucleus. These data reveal that both decapping enzymes are contained in the nuclear compartment, indicating that they may fulfill a greater function in the nucleus than previously appreciated.

Base Pairing↗

PubMed2002

The hDcp2 protein is a mammalian mRNA decapping enzyme.

Decapping of mRNA is a critical step in eukaryotic mRNA turnover, yet the proteins involved in this activity remain elusive in mammals. We identified the human Dcp2 protein (hDcp2) as an enzyme containing intrinsic decapping activity. hDcp2 specifically hydrolyzed methylated capped RNA to release m(7)GDP; however, it did not function on the cap structure alone. hDcp2 is therefore functionally distinct from the recently identified mammalian scavenger decapping enzyme, DcpS. hDcp2-mediated decapping required a functional Nudix (nucleotide diphosphate linked to an X moiety) pyrophosphatase motif as mutations in conserved amino acids within this motif disrupted the decapping activity. hDcp2 is detected exclusively in the cytoplasm and predominantly cosediments with polysomes. Consistent with the localization of hDcp2, endogenous Dcp2-like decapping activity was detected in polysomal fractions prepared from mammalian cells. Similar to decapping in yeast, the presence of the poly(A) tail was inhibitory to the endogenous decapping activity, yet unlike yeast, competition of cap-binding proteins by cap analog did not influence the efficiency of decapping. Therefore the mammalian homologue of the yeast Dcp2 protein is an mRNA decapping enzyme demonstrated to contain intrinsic decapping activity.

Amino Acid Motifs↗

PubMed2002

The scavenger mRNA decapping enzyme DcpS is a member of the HIT family of pyrophosphatases.

We recently demonstrated that the major decapping activity in mammalian cells involves DcpS, a scavenger pyrophosphatase that hydrolyzes the residual cap structure following 3' to 5' decay of an mRNA. The association of DcpS with 3' to 5' exonuclease exosome components suggests that these two activities are linked and there is a coupled exonucleolytic decay-dependent decapping pathway. We purified DcpS from mammalian cells and identified the cDNA encoding a novel 40 kDa protein possessing DcpS activity. Consistent with purified DcpS, the recombinant protein specifically hydrolyzed methylated cap analog but did not hydrolyze unmethylated cap analog nor did it function on intact capped RNA. Sequence alignments of DcpS from different organisms revealed the presence of a conserved hexapeptide, containing a histidine triad (HIT) sequence with three histidines separated by hydrophobic residues. Mutagenesis analysis revealed that the central histidine within the DcpS HIT motif is critical for decapping activity and defines the HIT motif as a new mRNA decapping domain, making DcpS the first member of the HIT family of proteins with a defined biological function.

Amino Acid Motifs↗

PubMed2002

Identifying mRNAs bound by RNA-binding proteins using affinity purification and differential display.

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.

Animals↗

PubMed2002

Identification of target messenger RNA substrates for the murine deleted in azoospermia-like RNA-binding protein.

The murine autosomal deleted in azoospermia-like protein (mDAZL) is a germ cell-restricted RNA-binding protein essential for sperm production. Homozygous disruption of the mDAZL gene results in the absence of germ cells beyond the spermatogonial stage. Progress into the function of DAZL in spermatogenesis has been hampered without identification of the cognate mRNA substrates that it binds to and regulates. Using the isolation of specific nucleic acids associated with proteins (SNAAP) technique recently developed in our lab, we identified mRNAs from testis that were specifically bound by mDAZL. One mRNA encoded the Tpx-1 protein, a testicular cell adhesion protein essential for the progression of spermatogenesis. A 26-nucleotide region necessary and sufficient to bind mDAZL was found within additional mRNAs isolated by the screen. These included mRNA encoding Pam, a protein associated with myc; GRSF1, an mRNA-binding protein involved in translation activation, and TRF2, a TATA box-binding protein-like protein involved in transcriptional regulation. Each mRNA containing the mDAZL binding site was specifically bound by mDAZL. A similar sequence is also present in the Cdc25A mRNA, a threonine/tyrosine phosphatase involved in cell cycle progression. The mDAZL and Cdc25A homologues are functionally linked in Drosophila and are necessary for spermatogenesis. Our demonstration that Tpx-1 and Cdc25A mRNAs are bound by mDAZL suggests that mDAZL regulates a subset of mRNAs necessary for germ cell development and cell cycle progression. Understanding how mDAZL regulates the target mRNAs will provide new insights into spermatogenesis, strategies for therapeutic intervention in azoospermic patients, and novel approaches for male contraception.

Animals↗