Cotranslational microRNA mediated messenger RNA destabilization.

MicroRNAs are small (22 nucleotide) regulatory molecules that play important roles in a wide variety of biological processes. These RNAs, which bind to targeted mRNAs via limited base pairing interactions, act to reduce protein production from those mRNAs. Considerable evidence indicates that miRNAs destabilize targeted mRNAs by recruiting enzymes that function in normal mRNA decay and mRNA degradation is widely thought to occur when mRNAs are in a ribosome free state.

A rapid, quantitative assay for direct detection of microRNAs and other small RNAs using splinted ligation.

This protocol describes a method that uses splinted ligation for in-solution, direct labeling of small RNAs from total RNA. The liquid phase hybridization method makes it possible to achieve sensitive, specific, and quantitative detection while eliminating a number of time-consuming and labor-intensive steps required for the standard Northern blot assay. The assay uses a small RNA-specific bridge oligonucleotide to form base pairs with the small RNA and a 5' end radiolabeled ligation oligonucleotide.

Dynamic regulation of alternative splicing by silencers that modulate 5' splice site competition.

Alternative splicing makes a major contribution to proteomic diversity in higher eukaryotes with approximately 70% of genes encoding two or more isoforms. In most cases, the molecular mechanisms responsible for splice site choice remain poorly understood. Here, we used a randomization-selection approach in vitro to identify sequence elements that could silence a proximal strong 5' splice site located downstream of a weakened 5' splice site.

Direct detection of small RNAs using splinted ligation.

This protocol describes a method for direct labeling and detection of small RNAs present in total RNA by splinted ligation. The assay uses a small RNA-specific bridge oligonucleotide to form base pairs with the small RNA and a 5'-end-radiolabeled ligation oligonucleotide. The captured small RNA is directly labeled by ligation.

A rapid, quantitative assay for direct detection of microRNAs and other small RNAs using splinted ligation.

The discovery and characterization of microRNAs (miRNAs) and other families of short RNAs has led to a rapid expansion of research directed at elucidating their expression patterns and regulatory functions. Here, we describe a convenient, sensitive, and straightforward method to detect and quantitate specific miRNA levels in unfractionated total RNA samples. The method, based on splinted ligation, does not require specialized equipment or any amplification step, and is significantly faster and more sensitive than Northern blotting.

Evidence that microRNAs are associated with translating messenger RNAs in human cells.

MicroRNAs (miRNAs) regulate gene expression post-transcriptionally by binding the 3' untranslated regions of target mRNAs. We examined the subcellular distribution of three miRNAs in exponentially growing HeLa cells and found that the vast majority are associated with mRNAs in polysomes. Several lines of evidence indicate that most of these mRNAs, including a known miRNA-regulated target (KRAS mRNA), are actively being translated.

Discriminatory RNP remodeling by the DEAD-box protein DED1.

DExH/D proteins catalyze NTP-driven rearrangements of RNA and RNA-protein complexes during most aspects of RNA metabolism. Although the vast majority of DExH/D proteins displays virtually no sequence-specificity when remodeling RNA complexes in vitro, the enzymes clearly distinguish between a large number of RNA and RNP complexes in a physiological context. It is unknown how this discrimination between potential substrates is achieved.

Protein displacement by DExH/D "RNA helicases" without duplex unwinding.

Members of the DExH/D superfamily of nucleic acid-activated nucleotide triphosphatases are essential for virtually all aspects of RNA metabolism, including pre-messenger RNA splicing, RNA interference, translation, and nucleocytoplasmic trafficking. Physiological substrates for these enzymes are thought to be regions of double-stranded RNA, because several DExH/D proteins catalyze strand separation in vitro. These "RNA helicases" can also disrupt RNA-protein interactions, but it is unclear whether this activity is coupled to duplex unwinding.

New components of the spliced leader RNP required for nematode trans-splicing.

Pre-messenger-RNA maturation in nematodes and in several other lower eukaryotic phyla involves spliced leader (SL) addition trans-splicing. In this unusual RNA processing reaction, a short common 5' exon, the SL, is affixed to the 5'-most exon of multiple pre-mRNAs. The nematode SL is derived from a trans-splicing-specific approximately 100-nucleotide RNA (SL RNA) that bears striking similarities to the cis-spliceosomal U small nuclear RNAs U1, U2, U4 and U5 (refs 3, 4); for example, the SL RNA functions only if it is assembled into an Sm small nuclear ribonucleoprotein (snRNP).