The Clothes Make the mRNA: Past and Present Trends in mRNP Fashion.

Throughout their lifetimes, messenger RNAs (mRNAs) associate with proteins to form ribonucleoproteins (mRNPs). Since the discovery of the first mRNP component more than 40 years ago, what is known as the mRNA interactome now comprises >1,000 proteins. These proteins bind mRNAs in myriad ways with varying affinities and stoichiometries, with many assembling onto nascent RNAs in a highly ordered process during transcription and precursor mRNA (pre-mRNA) processing. The nonrandom distribution of major mRNP proteins observed in transcriptome-wide studies leads us to propose that mRNPs are organized into three major domains loosely corresponding to 5' untranslated regions (UTRs), open reading frames, and 3' UTRs. Moving from the nucleus to the cytoplasm, mRNPs undergo extensive remodeling as they are first acted upon by the nuclear pore complex and then by the ribosome. When not being actively translated, cytoplasmic mRNPs can assemble into large multi-mRNP assemblies or be permanently disassembled and degraded. In this review, we aim to give the reader a thorough understanding of past and current eukaryotic mRNP research.

UPF1 helicase promotes TSN-mediated miRNA decay.

While microRNAs (miRNAs) regulate the vast majority of protein-encoding transcripts, little is known about how miRNAs themselves are degraded. We recently described Tudor-staphylococcal/micrococcal-like nuclease (TSN)-mediated miRNA decay (TumiD) as a cellular pathway in which the nuclease TSN promotes the decay of miRNAs that contain CA and/or UA dinucleotides. While TSN-mediated degradation of either protein-free or AGO2-loaded miRNAs does not require the ATP-dependent RNA helicase UPF1 in vitro, we report here that cellular TumiD requires UPF1. Results from experiments using AGO2-loaded miRNAs in duplex with target mRNAs indicate that UPF1 can dissociate miRNAs from their mRNA targets, making the miRNAs susceptible to TumiD. miR-seq (deep sequencing of miRNAs) data reveal that the degradation of ∼50% of candidate TumiD targets in T24 human urinary bladder cancer cells is augmented by UPF1. We illustrate the physiological relevance by demonstrating that UPF1-augmented TumiD promotes the invasion of T24 cells in part by degrading anti-invasive miRNAs so as to up-regulate the expression of proinvasive proteins.

Substrate-assisted mechanism of RNP disruption by the spliceosomal Brr2 RNA helicase.

The Brr2 RNA helicase disrupts the U4/U6 di-small nuclear RNA-protein complex (di-snRNP) during spliceosome activation via ATP-driven translocation on the U4 snRNA strand. However, it is unclear how bound proteins influence U4/U6 unwinding, which regions of the U4/U6 duplex the helicase actively unwinds, and whether U4/U6 components are released as individual molecules or as subcomplexes. Here, we set up a recombinant Brr2-mediated U4/U6 di-snRNP disruption system, showing that sequential addition of the U4/U6 proteins small nuclear ribonucleoprotein-associated protein 1 (Snu13), pre-mRNA processing factor 31 (Prp31), and Prp3 to U4/U6 di-snRNA leads to a stepwise decrease of Brr2-mediated U4/U6 unwinding, but that unwinding is largely restored by a Brr2 cofactor, the C-terminal Jab1/MPN domain of the Prp8 protein. Brr2-mediated U4/U6 unwinding was strongly inhibited by mutations in U4/U6 di-snRNAs that diminish the ability of U6 snRNA to adopt an alternative conformation but leave the number and kind of U4/U6 base pairs unchanged. Irrespective of the presence of the cofactor, the helicase segregated a Prp3-Prp31-Snu13-U4/U6 RNP into an intact Prp31-Snu13-U4 snRNA particle, free Prp3, and free U6 snRNA. Together, these observations suggest that Brr2 translocates only a limited distance on the U4 snRNA strand and does not actively release RNA-bound proteins. Unwinding is then completed by the partially displaced U6 snRNA adopting an alternative conformation, which leads to dismantling of the Prp3-binding site on U4/U6 di-snRNA but leaves the Prp31- and Snu13-binding sites on U4 snRNA unaffected. In this fashion, Brr2 can activate the spliceosome by stripping U6 snRNA of all precatalytic binding partners, while minimizing logistic requirements for U4/U6 di-snRNP reassembly after splicing.