RNA stabilization by a poly(A) tail 3'-end binding pocket and other modes of poly(A)-RNA interaction.

Polyadenylate [poly(A)] tail addition to the 3' end of a wide range of RNAs is a highly conserved modification that plays a central role in cellular RNA function. Elements for nuclear expression (ENEs) are cis-acting RNA elements that stabilize poly(A) tails by sequestering them in RNA triplex structures. A crystal structure of a double ENE from a rice hAT transposon messenger RNA complexed with poly(A) at a resolution of 2.

Fluorescence Amplification Method for Forward Genetic Discovery of Factors in Human mRNA Degradation.

Nonsense-mediated decay (NMD) degrades mRNAs containing a premature termination codon (PTC). PTCs are a frequent cause of human genetic diseases, and the NMD pathway is known to modulate disease severity. Since partial NMD attenuation can potentially enhance nonsense suppression therapies, better definition of human-specific NMD is required.

A proximity-dependent assay for specific RNA-protein interactions in intact cells.

The proximity ligation assay (PLA) is an immune staining method that detects protein-protein interactions in fixed cells. We describe here RNA-PLA, a simple adaptation of this technology that allows the detection of specific RNA-protein interactions in fixed cells by using a DNA oligonucleotide that hybridizes to a target RNA in combination with an antibody that recognizes the protein bound to the target RNA. Stable and transient RNA-protein interactions can be readily detected by generation of a fluorescent signal in discrete compartments in intact fixed cells with high specificity.

Myriad Triple-Helix-Forming Structures in the Transposable Element RNAs of Plants and Fungi.

The ENE (element for nuclear expression) is a cis-acting RNA structure that protects viral or cellular noncoding RNAs (ncRNAs) from nuclear decay through triple-helix formation with the poly(A) tail or 3'-terminal A-rich tract. We expanded the roster of nine known ENEs by bioinformatic identification of ∼200 distinct ENEs that reside in transposable elements (TEs) of numerous non-metazoan and one fish species and in four Dicistrovirus genomes. Despite variation within the ENE core, none of the predicted triple-helical stacks exceeds five base triples.

The host Integrator complex acts in transcription-independent maturation of herpesvirus microRNA 3' ends.

Herpesvirus saimiri (HVS) is an oncogenic γ-herpesvirus that produces microRNAs (miRNAs) by cotranscription of precursor miRNA (pre-miRNA) hairpins immediately downstream from viral small nuclear RNAs (snRNA). The host cell Integrator complex, which recognizes the snRNA 3' end processing signal (3' box), generates the 5' ends of HVS pre-miRNA hairpins. Here, we identify a novel 3' box-like sequence (miRNA 3' box) downstream from HVS pre-miRNAs that is essential for miRNA biogenesis.

Mammalian 5'-capped microRNA precursors that generate a single microRNA.

MicroRNAs (miRNAs) are short RNA gene regulators typically produced from primary transcripts that are cleaved by the nuclear microprocessor complex, with the resulting precursor miRNA hairpins exported by exportin 5 and processed by cytoplasmic Dicer to yield two (5p and 3p) miRNAs. Here, we document microprocessor-independent 7-methylguanosine (m(7)G)-capped pre-miRNAs, whose 5' ends coincide with transcription start sites and 3' ends are most likely generated by transcription termination. By establishing a small RNA Cap-seq method that employs the cap-binding protein eIF4E, we identified a group of murine m(7)G-capped pre-miRNAs genome wide.

Human spliceosomal protein CWC22 plays a role in coupling splicing to exon junction complex deposition and nonsense-mediated decay.

The multiprotein exon junction complex (EJC) that is deposited upstream of spliced junctions orchestrates downstream events in the life of a metazoan mRNA, including its surveillance via the nonsense-mediated decay (NMD) pathway. However, the mechanism by which the spliceosome mediates EJC formation is not well understood. We show that human eIF4G-like spliceosomal protein (h)CWC22 directly interacts with the core EJC component eIF4AIII in vitro and in vivo; mutations at the predicted hCWC22/eIF4AIII interface disrupt association.

Conservation of a triple-helix-forming RNA stability element in noncoding and genomic RNAs of diverse viruses.

Abundant expression of the long noncoding (lnc) PAN (polyadenylated nuclear) RNA by the human oncogenic gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) depends on a cis-element called the expression and nuclear retention element (ENE). The ENE upregulates PAN RNA by inhibiting its rapid nuclear decay through triple-helix formation with the poly(A) tail. Using structure-based bioinformatics, we identified six ENE-like elements in evolutionarily diverse viral genomes.

A conserved WD40 protein binds the Cajal body localization signal of scaRNP particles.

Small Cajal body (CB)-specific RNPs (scaRNPs) function in posttranscriptional modification of small nuclear (sn)RNAs. An RNA element, the CAB box, facilitates CB localization of H/ACA scaRNPs. Using a related element in Drosophila C/D scaRNAs, we purified a fly WD40 repeat protein that UV crosslinks to RNA in a C/D CAB box-dependent manner and associates with C/D and mixed domain C/D-H/ACA scaRNAs.

Minor-class splicing occurs in the nucleus of the Xenopus oocyte.

A small fraction of premessenger RNA introns in certain eukaryotes is excised by the minor spliceosome, which contains low-abundance small nuclear ribonucleoproteins (snRNPs). Recently, it was suggested that minor-class snRNPs are localized to and function in the cytoplasm of vertebrate cells. To test whether U12-type splicing occurs in the cytoplasm of Xenopus oocytes, we performed microinjections of the well-characterized P120 minor-class splicing substrate into the nucleus or into the cytoplasm.

Mutational analysis of a viral RNA element that counteracts rapid RNA decay by interaction with the polyadenylate tail.

We previously demonstrated that the Kaposi's sarcoma-associated herpesvirus polyadenylated nuclear RNA contains a 79-nt cis-acting element, the ENE, which allows intronless polyadenylated transcripts to accumulate to high nuclear levels by protecting them from rapid degradation. We proposed a model based on the predicted structure of the ENE in which a U-rich internal loop hybridizes with the 3'-polyadenylate (polyA) tail to sequester it from exonucleolytic attack. We have tested this model by mutational analysis of the ENE.

Identification of a rapid mammalian deadenylation-dependent decay pathway and its inhibition by a viral RNA element.

Cellular RNAs are subject to quality-control pathways that insure the fidelity of gene expression. We previously identified a 79 nt element, the ENE, that is essential for the nuclear accumulation of a viral polyadenylated nuclear (PAN) RNA. Here, we show that intron-less polyadenylated transcripts such as PAN RNA and beta-globin cRNA exhibit two-component exponential decay kinetics in which some transcripts are rapidly degraded (t(1/2) = approximately 15 min) while others decay more slowly (t(1/2) = approximately 3 hr).

A spliceosomal intron binding protein, IBP160, links position-dependent assembly of intron-encoded box C/D snoRNP to pre-mRNA splicing.

Pre-mRNA splicing in vertebrates is molecularly linked to other processes. We previously reported that splicing is required for efficient assembly of intron-encoded box C/D small nucleolar ribonucleoprotein (snoRNP). In the spliceosomal C1 complex, snoRNP proteins efficiently assemble onto snoRNA sequences if they are located about 50 nt upstream of the intron branchpoint.

Splicing of U12-type introns deposits an exon junction complex competent to induce nonsense-mediated mRNA decay.

Metazoan cells have two pathways for intron removal involving the U2- and U12-type spliceosomes, which contain mostly nonoverlapping sets of small nuclear ribonucleoproteins. We show that in vitro splicing of a U12-type intron assembles an exon junction complex (EJC) that is comparably positioned and contains many of the same components as that deposited by the U2-type spliceosome. The presence of a U12-type intron downstream of a premature termination codon within an open reading frame (ORF) induces nonsense-mediated decay of the mRNA in vivo.

Splicing-dependent and -independent modes of assembly for intron-encoded box C/D snoRNPs in mammalian cells.

In mammalian cells, all small nucleolar RNAs (snoRNAs) that guide rRNA modification are encoded within the introns of host genes. An optimal position about 70 nts upstream of the 3' splice site of the host intron is critical for efficient expression of box C/D snoRNAs in vivo, suggesting synergy with splicing. Here, we have used a coupled in vitro splicing-snoRNA processing system to demonstrate that assembly of box C/D snoRNP proteins is the step affected by snoRNA location, and that active splicing is essential for snoRNP assembly.