Human monoclonal antibody MBL-HCV1 delays HCV viral rebound following liver transplantation: a randomized controlled study.

Rapid allograft infection complicates liver transplantation (LT) in patients with hepatitis C virus (HCV). Pegylated interferon-α and ribavirin therapy after LT has significant toxicity and limited efficacy. The effect of a human monoclonal antibody targeting the HCV E2 glycoprotein (MBL-HCV1) on viral clearance was examined in a randomized, double-blind, placebo-controlled pilot study in patients infected with HCV genotype 1a undergoing LT. Subjects received 11 infusions of 50 mg/kg MBL-HCV1 (n=6) or placebo (n=5) intravenously with three infusions on day of transplant, a single infusion on days 1 through 7 and one infusion on day 14 after LT. MBL-HCV1 was well-tolerated and reduced viral load for a period ranging from 7 to 28 days. Median change in viral load (log10 IU/mL) from baseline was significantly greater (p=0.02) for the antibody-treated group (range -3.07 to -3.34) compared to placebo group (range -0.331 to -1.01) on days 3 through 6 posttransplant. MBL-HCV1 treatment significantly delayed median time to viral rebound compared to placebo treatment (18.7 days vs. 2.4 days, p<0.001). As with other HCV monotherapies, antibody-treated subjects had resistance-associated variants at the time of viral rebound. A combination study of MBL-HCV1 with a direct-acting antiviral is underway.

MicroRNA therapeutics.

MicroRNAs (miRNAs) provide new therapeutic targets for many diseases, while their myriad roles in development and cellular processes make them fascinating to study. We still do not fully understand the molecular mechanisms by which miRNAs regulate gene expression nor do we know the complete repertoire of mRNAs each miRNA regulates. However, recent progress in the development of effective strategies to block miRNAs suggests that anti-miRNA drugs may soon be used in the clinic.

Therapeutic silencing of mutant huntingtin with siRNA attenuates striatal and cortical neuropathology and behavioral deficits.

Huntington's disease (HD) is a neurodegenerative disorder caused by expansion of a CAG repeat in the huntingtin (Htt) gene. HD is autosomal dominant and, in theory, amenable to therapeutic RNA silencing. We introduced cholesterol-conjugated small interfering RNA duplexes (cc-siRNA) targeting human Htt mRNA (siRNA-Htt) into mouse striata that also received adeno-associated virus containing either expanded (100 CAG) or wild-type (18 CAG) Htt cDNA encoding huntingtin (Htt) 1-400. Adeno-associated virus delivery to striatum and overlying cortex of the mutant Htt gene, but not the wild type, produced neuropathology and motor deficits. Treatment with cc-siRNA-Htt in mice with mutant Htt prolonged survival of striatal neurons, reduced neuropil aggregates, diminished inclusion size, and lowered the frequency of clasping and footslips on balance beam. cc-siRNA-Htt was designed to target human wild-type and mutant Htt and decreased levels of both in the striatum. Our findings indicate that a single administration into the adult striatum of an siRNA targeting Htt can silence mutant Htt, attenuate neuronal pathology, and delay the abnormal behavioral phenotype observed in a rapid-onset, viral transgenic mouse model of HD.

Measuring the rates of transcriptional elongation in the female Drosophila melanogaster germ line by nuclear run-on.

We adapted the nuclear run-on method to measure changes in the rate of RNA polymerase II (pol II) transcription of repetitive elements and transposons in the female germ line of Drosophila melanogaster. Our data indicate that as little as an approximately 1.5-fold change in the rate of transcription can be detected by this method. Our nuclear run-on protocol likely measures changes in transcriptional elongation, because rates of transcription decline with time, consistent with a low rate of pol II re-initiation in the isolated nuclei. Surprisingly, we find that the retrotransposon gypsy and the repetitive sequence mst40 are silenced posttranscriptionally in fly ovaries.

Comparative analysis of the plant mRNA-destabilizing element, DST, in mammalian and tobacco cells.

The labile SAUR transcripts from higher plants contain a conserved DST sequence in their 3'-untranslated regions. Two copies of a DST sequence from soybean are sufficient to destabilize reporter transcripts in cultured tobacco cells whereas variants bearing mutations in the conserved ATAGAT or GTA regions are inactive. To investigate the potential for conserved recognition components in mammalian and plant cells, we examined the function of this instability determinant in mouse NIH3T3 fibroblasts and tobacco BY2 cells. In fibroblasts, a tetrameric DST element from soybean accelerated deadenylation and decay of a reporter transcript. However, a version mutated in the ATAGAT region was equally effective in this regard, and a tetrameric DST element from Arabidopsis was inactive. In contrast, the soybean DST element was more active as an mRNA instability element than the mutant version and the Arabidopsis element, when tested as tetramers in tobacco cells. Hence, the plant DST element is not recognized in animal cells with the same sequence requirements as in plant cells. Therefore, its mode of recognition appears to be plant-specific.

ATP requirements and small interfering RNA structure in the RNA interference pathway.

We examined the role of ATP in the RNA interference (RNAi) pathway. Our data reveal two ATP-dependent steps and suggest that the RNAi reaction comprises at least four sequential steps: ATP-dependent processing of double-stranded RNA into small interfering RNAs (siRNAs), incorporation of siRNAs into an inactive approximately 360 kDa protein/RNA complex, ATP-dependent unwinding of the siRNA duplex to generate an active complex, and ATP-independent recognition and cleavage of the RNA target. Furthermore, ATP is used to maintain 5' phosphates on siRNAs. A 5' phosphate on the target-complementary strand of the siRNA duplex is required for siRNA function, suggesting that cells check the authenticity of siRNAs and license only bona fide siRNAs to direct target RNA destruction.

RNA interference: listening to the sound of silence.

The term RNA interference (RNAi) describes the use of double-stranded RNA to target specific mRNAs for degradation, thereby silencing their expression. RNAi is one manifestation of a broad class of RNA silencing phenomena that are found in plants, animals and fungi. The discovery of RNAi has changed our understanding of how cells guard their genomes, led to the development of new strategies for blocking gene function, and may yet yield RNA-based drugs to treat human disease.

A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA.

The 21-nucleotide small temporal RNA (stRNA) let-7 regulates developmental timing in Caenorhabditis elegans and probably in other bilateral animals. We present in vivo and in vitro evidence that in Drosophila melanogaster a developmentally regulated precursor RNA is cleaved by an RNA interference-like mechanism to produce mature let-7 stRNA. Targeted destruction in cultured human cells of the messenger RNA encoding the enzyme Dicer, which acts in the RNA interference pathway, leads to accumulation of the let-7 precursor. Thus, the RNA interference and stRNA pathways intersect. Both pathways require the RNA-processing enzyme Dicer to produce the active small-RNA component that represses gene expression.

Crystal structure of a Pumilio homology domain.

Puf proteins regulate translation and mRNA stability by binding sequences in their target RNAs through the Pumilio homology domain (PUM-HD), which is characterized by eight tandem copies of a 36 amino acid motif, the PUM repeat. We have solved the structure of the PUM-HD from human Pumilio1 at 1.9 A resolution. The structure reveals that the eight PUM repeats correspond to eight copies of a single, repeated structural motif. The PUM repeats pack together to form a right-handed superhelix that approximates a half doughnut. The distribution of side chains on the inner and outer faces of this half doughnut suggests that the inner face of the PUM-HD binds RNA while the outer face interacts with proteins such as Nanos, Brain Tumor, and cytoplasmic polyadenylation element binding protein.

Thirty-three years later, a glimpse at the ribonuclease III active site.

RNase III endonucleases cleave double-stranded RNA, transforming precursor RNAs into mature RNAs that act in pre-mRNA splicing, RNA modification, translation, gene silencing, and the regulation of developmental timing. The recently solved structure of an RNase III endonuclease domain provides a hint at how this family of ribonucleases functions.

RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals.

Double-stranded RNA (dsRNA) directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). Using a recently developed Drosophila in vitro system, we examined the molecular mechanism underlying RNAi. We find that RNAi is ATP dependent yet uncoupled from mRNA translation. During the RNAi reaction, both strands of the dsRNA are processed to RNA segments 21-23 nucleotides in length. Processing of the dsRNA to the small RNA fragments does not require the targeted mRNA. The mRNA is cleaved only within the region of identity with the dsRNA. Cleavage occurs at sites 21-23 nucleotides apart, the same interval observed for the dsRNA itself, suggesting that the 21-23 nucleotide fragments from the dsRNA are guiding mRNA cleavage.

The PUMILIO-RNA interaction: a single RNA-binding domain monomer recognizes a bipartite target sequence.

Translational repression of hunchback (hb) mRNA in the posterior of the Drosophila embryo requires two copies of a bipartite sequence, the Nanos Response Element (NRE), located in the 3' untranslated region of the mRNA. The PUMILIO (PUM) protein is thought to bind the NREs and thereby repress hb translation. The RNA-binding domain of PUM defines an evolutionarily conserved family of RNA-binding proteins, the PUM-Homology Domain (PUM-HD) proteins, which have been identified in yeast, plants, and animals. The PUM RNA-binding domain, the Drosophila PUM-HD (DmPUM-HD), has been shown previously to recognize nucleotides in both the 5' and 3' halves of the NRE, suggesting that a dimer of PUM might recognize one NRE. Here, we analyze the RNA-binding affinity and stoichiometry of the DmPUM-HD and find that one DmPUM-HD monomer binds independently and with equal affinity to each NRE (KD approximately 0.5 nM). We detect no cooperative interactions between DmPUM-HD monomers bound at adjacent sites. Our results imply that a single DmPUM-HD protein recognizes nucleotides in both the 5' and 3' NRE half-sites. Based on our estimate of the intraembryonic concentration of PUM (>40 nM), we propose that in vivo nearly all NREs are occupied by a PUM monomer.

Targeted mRNA degradation by double-stranded RNA in vitro.

Double-stranded RNA (dsRNA) directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function. The biochemical mechanisms underlying this dsRNA interference (RNAi) are unknown. Here we report the development of a cell-free system from syncytial blastoderm Drosophila embryos that recapitulates many of the features of RNAi. The interference observed in this reaction is sequence specific, is promoted by dsRNA but not single-stranded RNA, functions by specific mRNA degradation, and requires a minimum length of dsRNA. Furthermore, preincubation of dsRNA potentiates its activity. These results demonstrate that RNAi can be mediated by sequence-specific processes in soluble reactions.

The Pumilio protein binds RNA through a conserved domain that defines a new class of RNA-binding proteins.

Translation of hunchback(mat) (hb[mat]) mRNA must be repressed in the posterior of the pre-blastoderm Drosophila embryo to permit formation of abdominal segments. This translational repression requires two copies of the Nanos Response Element (NRE), a 16-nt sequence in the hb[mat] 3' untranslated region. Translational repression also requires the action of two proteins: Pumilio (PUM), a sequence-specific RNA-binding protein; and Nanos, a protein that determines the location of repression. Binding of PUM to the NRE is thought to target hb(mat) mRNA for repression. Here, we show the RNA-binding domain of PUM to be an evolutionarily conserved, 334-amino acid region at the carboxy-terminus of the approximately 158-kDa PUM protein. This contiguous region of PUM retains the RNA-binding specificity of full-length PUM protein. Proteins with sequences homologous to the PUM RNA-binding domain are found in animals, plants, and fungi. The high degree of sequence conservation of the PUM RNA-binding domain in other far-flung species suggests that the domain is an ancient protein motif, and we show that conservation of sequence reflects conservation of function: that is, the homologous region from a human protein binds RNA with sequence specificity related to but distinct from Drosophila PUM.

A CCHC metal-binding domain in Nanos is essential for translational regulation.

The Drosophila Nanos protein is a localized repressor of hunchback mRNA translation in the early embryo, and is required for the establishment of the anterior-posterior body axis. Analysis of nanos mutants reveals that a small, evolutionarily conserved, C-terminal region is essential for Nanos function in vivo, while no other single portion of the Nanos protein is absolutely required. Within the C-terminal region are two unusual Cys-Cys-His-Cys (CCHC) motifs that are potential zinc-binding sites. Using absorption spectroscopy and NMR we demonstrate that the CCHC motifs each bind one equivalent of zinc with high affinity. nanos mutations disrupting metal binding at either of these two sites in vitro abolish Nanos translational repression activity in vivo. We show that full-length and C-terminal Nanos proteins bind to RNA in vitro with high affinity, but with little sequence specificity. Mutations affecting the hunchback mRNA target sites for Nanos-dependent translational repression were found to disrupt translational repression in vivo, but had little effect on Nanos RNA binding in vitro. Thus, the Nanos zinc domain does not specifically recognize target hunchback RNA sequences, but might interact with RNA in the context of a larger ribonucleoprotein complex.

Drosophila development: homeodomains and translational control.

Translation of the transcription factor caudal is repressed at the anterior end of the Drosophila embryo. Surprisingly, the DNA-binding homeodomain of the transcription factor Bicoid mediates this repression by binding caudal mRNA.

RNA annealing activity is intrinsically associated with U2AF.

U2AF is a protein that is essential for the formation of the prespliceosome complex during pre-mRNA splicing. It contains two subunits, 65 and 35 kDa, although only the 65-kDa subunit has been shown to be essential for its splicing activity. Here, we show that the 65-kDa subunit mediates the annealing of complementary single-stranded RNAs or single-stranded DNAs. This activity was shown to reverse the action of RNA helicase A, an enzyme that catalyzes the displacement of duplex RNAs. The NH2-terminal region of the 65-kDa subunit of U2AF, containing arginine-serine (RS) dipeptides and basic amino acid sequences, was shown to be essential for the annealing of complementary sequences, RNA binding, and the inhibition of RNA helicase A activity. Thus, through the combined action of U2AF and RNA helicases, duplex RNA regions can be reversibly formed and displaced. Such reactions appear to be critical for pre-mRNA splicing, translation, and transcription.