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Cis-acting RNA elements are crucial for the regulation of polyadenylated RNA stability. The element for nuclear expression (ENE) contains a U-rich internal loop flanked by short helices. An ENE stabilizes RNA by sequestering the poly(A) tail via formation of a triplex structure that inhibits a rapid deadenylation-dependent decay pathway. Structure-based bioinformatic studies identified numerous ENE-like elements in evolutionarily diverse genomes, including a subclass containing two ENE motifs separated by a short double-helical region (double ENEs [dENEs]). Here, the structure of a dENE derived from a rice transposable element (TWIFB1) before and after poly(A) binding (~24 kDa and ~33 kDa, respectively) is investigated. We combine biochemical structure probing, small angle X-ray scattering (SAXS), and cryo-electron microscopy (cryo-EM) to investigate the dENE structure and its local and global structural changes upon poly(A) binding. Our data reveal 1) the directionality of poly(A) binding to the dENE, and 2) that the dENEpoly(A) interaction involves a motif that protects the 3'-most seven adenylates of the poly(A). Furthermore, we demonstrate that the dENE does not undergo a dramatic global conformational change upon poly(A) binding. These findings are consistent with the recently solved crystal structure of a dENE+poly(A) complex [S.-F. Torabi et al., Science 371, eabe6523 (2021)]. Identification of additional modes of poly(A)-RNA interaction opens new venues for better understanding of poly(A) tail biology. [ABSTRACT FROM AUTHOR]

Hfq is a small, highly abundant hexameric protein that is found in many bacteria and plays a critical role in mRNA expression and RNA stability. As an "RNA chaperone," Hfq binds AU-rich sequences and facilitates the trans annealing of small RNAs (sRNAs) to their target mRNAs, typically resulting in the down-regulation of gene expression. Hfq also plays a key role in bacterial RNA decay by binding tightly to polyadenylate [poly(A)] tracts. The structural mechanism by which Hfq recognizes and binds poly(A) is unknown. Here, we report the crystal structure of Escherichia coil Hfq bound to the poly(A) RNA, A15. The structure reveals a unique RNA binding mechanism. Unlike uridine-containing sequences, which bind to the "proximal" face, the poly(A) tract binds to the "distal" face of Hfq using 6 tripartite binding motifs. Each motif consists of an adenosine specificity site (A site), which is effected by peptide backbone hydrogen bonds, a purine nucleotide selectivity site (R site), and a sequence-nondiscriminating RNA entrance/exit site (E site). The resulting implication that Hfq can bind poly(A-R-N) triplets, where R is a purine nucleotide and N is any nucleotide, was confirmed by binding studies. Indeed, Hfq bound to the oligoribonucleotides (AGG)8, (AGC)8, and the shorter (A-R-N)4 sequence, AACAACAAGAAG, with nanomolar affinities. The abundance of (A-R-N)4 and (A-R-N)5 triplet repeats in the E. coil genome suggests additional RNA targets for Hfq. Further, the structure provides insight into Hfq-mediated sRNA-mRNA annealing and the role of Hfq in RNA decay. [ABSTRACT FROM AUTHOR]

The yeast chromatin protein Sin1p/Spt2p has long been studied, but the understanding of its function has remained elusive. The protein has sequence similarity to HMG1, specifically binds crossing DNA structures, and serves as a negative transcriptional regulator of a small family of genes that are activated by the SWI/SNF chromatin-remodeling complex. Recently, it has been implicated in maintaining the integrity of chromatin during transcription elongation. Here we present experiments whose results indicate that Sin1p/Spt2 is required for, and is directly involved in, the efficient recruitment of the mRNA cleavage/polyadenylation complex. This conclusion is based on the following findings: Sin1p/Spt2 frequently binds specifically downstream of many ORFs but almost always upstream of the first polyadenylation site. It directly interacts with Fir1p, a component of the cleavage/polyadenylation complex. Disruption of Sin1p/Spt2p results in foreshortened poly(A) tracts on mRNA. It is synthetically lethal with Cdc73p, which is involved in the recruitment of the complex. This report shows that a chromatin component is involved in 3′ end processing of RNA. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are ubiquitous regulators of eukaryotic gene expression. In addition to repressing translation, miRNAs can down-regulate the concentration of mRNAs that contain elements to which they are imperfectly complementary. Using miR-125b and let-7 as representative miRNAs, we show that in mammalian cells this reduction in message abundance is a consequence of accelerated deadenylation, which leads to rapid mRNA decay. The ability of miRNAs to expedite poly(A) removal does not result from decreased translation; nor does translational repression by miRNAs require a poly(A) tail, a 3′ histone stem-loop being an effective substitute. These findings suggest that miRNAs use two distinct posttranscriptional mechanisms to down-regulate gene expression. [ABSTRACT FROM AUTHOR]