Jeffrey Wilusz

The mammalian exosome mediates the efficient degradation of mRNAs that contain AU‐rich elements

HeLa cytoplasmic extracts contain both 3′–5′ and 5′–3′ exonuclease activities that may play important roles in mRNA decay. Using an in vitro RNA deadenylation/decay assay, mRNA decay intermediates were trapped using phosphothioate‐modified RNAs. These data indicate that 3′–5′ exonucleolytic decay is the major pathway of RNA degradation following deadenylation in HeLa cytoplasmic extracts. Immunodepletion using antibodies specific for the exosomal protein PM‐Scl75 demonstrated that the human exosome complex is required for efficient 3′–5′ exonucleolytic decay. Furthermore, 3′–5′ exonucleolytic decay was stimulated dramatically by AU‐rich instability elements (AREs), implicating a role for the exosome in the regulation of mRNA turnover. Finally, PM‐Scl75 protein was found to interact specifically with AREs. These data suggest that the interaction between the exosome and AREs plays a key role in regulating the efficiency of ARE‐containing mRNA turnover.

A novel mRNA-decapping activity in HeLa cytoplasmic extracts is regulated by AU-rich elements

While decapping plays a major role in mRNA turnover in yeast, biochemical evidence for a similar activity in mammalian cells has been elusive. We have now identified a decapping activity in HeLa cytoplasmic extracts that releases 7meGDP from capped transcripts. Decapping is activated in extracts by the addition of 7meGpppG, which specifically sequesters cap‐binding proteins such as eIF4E and the deadenylase DAN/PARN. Similar to in vivo observations, the presence of a poly(A) tail represses decapping of RNAs in vitro in a poly(A)‐binding protein‐dependent fashion. AU‐rich elements (AREs), which act as regulators of mRNA stability in vivo, are potent stimulators of decapping in vitro. The stimulation of decapping by AREs requires sequence‐specific ARE‐binding proteins. These data suggest that cap recognition and decapping play key roles in mediating mRNA turnover in mammalian cells.

hnRNP F influences binding of a 64-kilodalton subunit of cleavage stimulation factor to mRNA precursors in mouse B cells.

ABSTRACT Previous studies on the regulation of polyadenylation of the immunoglobulin (Ig) heavy-chain pre-mRNA argued fortrans-acting modifiers of the cleavage-polyadenylation reaction operating differentially during B-cell developmental stages. Using four complementary approaches, we demonstrate that a change in the level of hnRNP F is an important determinant in the regulated use of alternative polyadenylation sites between memory and plasma stage B cells. First, by Western analyses of cellular proteins, the ratio of hnRNP F to H or H′ was found to be higher in memory B cells than in plasma cells. In memory B cells the activity of CstF-64 binding to pre-mRNA, but not its amount, was reduced. Second, examination of the complexes formed on input pre-mRNA in nuclear extracts revealed large assemblages containing hnRNP H, H′, and F but deficient in CstF-64 in memory B-cell extracts but not in plasma cells. Formation of these large complexes is dependent on the region downstream of the AAUAAA in pre-mRNA, suggesting that CstF-64 and the hnRNPs compete for a similar region. Third, using a recombinant protein we showed that hnRNP F could bind to the region downstream of a poly(A) site, block CstF-64 association with RNA, and inhibit the cleavage reaction. Fourth, overexpression of recombinant hnRNP F in plasma cells resulted in a decrease in the endogenous Ig heavy-chain mRNA secretory form-to-membrane ratio. These results demonstrate that mammalian hnRNP F can act as a negative regulator in the pre-mRNA cleavage reaction and that increased expression of F in memory B cells contributes to the suppression of the Ig heavy-chain secretory poly(A) site.

Serum-deprivation stimulates cap-binding by PARN at the expense of eIF4E, consistent with the observed decrease in mRNA stability

PARN, a poly(A)-specific ribonuclease, binds the 5′ cap-structure of mRNA and initiates deadenylation-dependent decay. Eukaryotic initiation factor 4E (eIF4E) also binds to the cap structure, an interaction that is critical for initiating cap-dependent translation. The stability of various mRNA transcripts in human cell lines is reduced under conditions of serum starvation as determined by both functional and chemical half-lives. Serum starvation also leads to enhanced cap association by PARN. In contrast, the 5′ cap occupancy by eIF4E decreases under serum-deprivation, as does the translation of reporter transcripts. Further, we show that PARN is a phosphoprotein and that this modification can be modulated by serum status. Taken together, these data are consistent with a natural competition existing at the 5′ cap structure between PARN and eIF4E that may be regulated by changes in post-translational modifications. These phosphorylation-induced changes in the interplay of PARN and eIF4E may determine whether the mRNA is translated or decayed.

The relationship between the prothrombin upstream sequence element and the G20210A polymorphism: the influence of a competitive environment for mRNA 3′-end formation

The human prothrombin G20210A polymorphism located at the 3′ cleavage site of the mRNA results in elevated plasma prothrombin levels and increased risk of venous thrombosis. This polymorphism has been shown to directly influence a variety of processes related to prothrombin mRNA metabolism. We have constructed plasmids that express the full-length prothrombin mRNA that is polyadenylated at its natural site. The A allele prothrombin variant was more efficient than the G allele at promoting cleavage at this site in the presence of a competing poly (A) sequence. In the absence of competition, both allelic variants give rise to a similar level of cleavage site heterogeneity. An upstream sequence element (USE) was also identified within the prothrombin 3′-UTR. When placed upstream of two competing poly (A) sites, the USE directed cleavage preferentially to the proximal poly (A) site. In the absence of competition, the USE had no effect on cleavage site selection. This study suggests that the basis for the increase in prothrombin expression in A allele carriers is not due to allelic changes in cleavage site selection per se. In addition, the functionality of USEs needs to be considered within the context of endogenous sequence architecture.

Rapid Identification and Cloning of Sequence-Specific RNA Binding Proteins

Publisher Summary This chapter presents a UV cross-linking approach to identify sequence-specific Ribonucleic acid (RNA) binding proteins in in vitro reconstitution assays. A typical result in the early stages in studies of messenger ribonucleic acid (mRNA) biology is the identification of a region of a transcript that plays a pivotal role in the control of a posttranscriptional process. The next key focus of the study is determining the mechanism by which this RNA signal functions. It is likely that many RNA signals work through the sequence- or structure-specific assembly of protein–RNA complexes. This chapter provides an overview of approaches used to identify the protein binding site on the transcript. These methodologies provide a reasonably rapid and efficient strategy for the identification and molecular characterization of RNA binding proteins. The chapter discusses in detail about the preparation of extracts and preparation of RNAs. Analysis of UV cross-linked protein-RNA complexes, identification of protein–RNA complexes, and determination of RNA sequence requirements for protein binding is also discussed in detail. The chapter also highlights cloning complementary Deoxyribonucleic acids (cDNAs) for RNA binding proteins by northwestern screening.