Polyadenylation is an essential step for the maturation of almost all cellular mRNAs in eukaryotes. In human cells, most poly(A) sites are flanked by the upstream AAUAAA hexamer or a close variant, and downstream U/GU-rich elements. In yeast and plants, additional cis elements have been found to be located upstream of the poly(A) site, including UGUA, UAUA, and U-rich elements. In this study, we have developed a computer program named PROBE (Polyadenylation-Related Oligonucleotide Bidimensional Enrichment) to identify cis elements that may play regulatory roles in mRNA polyadenylation. By comparing human genomic sequences surrounding frequently used poly(A) sites with those surrounding less frequently used ones, we found that cis elements occurring in yeast and plants also exist in human poly(A) regions, including the upstream U-rich elements, and UAUA and UGUA elements. In addition, several novel elements were found to be associated with human poly(A) sites, including several G-rich elements. Thus, we suggest that many cis elements are evolutionarily conserved among eukaryotes, and human poly(A) sites have an additional set of cis elements that may be involved in the regulation of mRNA polyadenylation.

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-modi®ed RNAs. These data indicate that 3¢±5¢ exonucleolytic decay is the major pathway of RNA degradation following deadenylation in HeLa cytoplasmic extracts. Immuno-depletion using antibodies speci®c for the exosomal protein PM-Scl75 demonstrated that the human exosome complex is required for ef®cient 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 speci®cally with AREs. These data suggest that the interaction between the exosome and AREs plays a key role in regulating the ef®ciency of ARE-containing mRNA turnover.

Adenosine receptor ligands have anti-inflammatory effects and modulate immune responses by up-regulating IL-10 production by immunostimulated macrophages. The adenosine receptor family comprises G protein-coupled heptahelical transmembrane receptors classified into four types: A1, A2A, A2B, and A3. Our understanding of the signaling mechanisms leading to enhanced IL-10 production following adenosine receptor occupancy on macrophages is limited. In this study, we demonstrate that adenosine receptor occupancy increases IL-10 production by LPS-stimulated macrophages without affecting IL-10 promoter activity and IL-10 mRNA levels, indicating a posttranscriptional mechanism. Transfection experiments with reporter constructs containing sequences corresponding to the AU-rich 3′-untranslated region (UTR) of IL-10 mRNA confirmed that adenosine receptor activation acts by relieving the translational repressive effect of the IL-10 3′-UTR. By contrast, adenosine receptor activation failed to liberate the translational arrest conferred by the 3′-UTR of TNF-α mRNA. The IL-10 3′-UTR formed specific complexes with proteins present in cytoplasmic extracts of RAW 264.7 cells. Adenosine enhanced binding of proteins to a region of the IL-10 3′-UTR containing the GUAUUUAUU nonamer. The stimulatory effect of adenosine on IL-10 production was mediated through the A2B receptor, because the order of potency of selective agonists was 5′-N-ethylcarboxamidoadenosine (NECA) > N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (IB-MECA) > 2-chloro-N6-cyclopentyladenosine (CCPA) = 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethyl-carboxamidoadenosine (CGS-21680). Also, the selective A2B antagonist, alloxazine, prevented the effect of adenosine. Collectively, these studies identify a novel pathway in which activation of a G protein-coupled receptor augments translation of an anti-inflammatory gene.

An integral component of the splicing machinery, the Ul snRNP, is here implicated in the efficient polyadenylation of SV40 late mRNAs. This occurs as a result of an interaction between Ul snRNP-A protein and the upstream efficiency element (USE) of the polyadenylation signal. UV cross-linking and immunoprecipitation demonstrate that this interaction can occur while Ul snRNP-A protein is simultaneously bound to Ul RNA as part of the snRNP. The dual reactivity of Ul snRNP-A occurs because the protein has two RNA recognition motifs (RRMs). The target RNA of the first RRM (RRMl) has been shown previously to be the second stem-loop of Ul RNA. We have found that a target for the second RRM (RRM2) is within the AUUUGURA motifs of the USE of the SV40 late polyadenylation signal. RNA substrates containing the wild-type USE efficiently bind to Ul snRNP-A protein, whereas substrates fail to bind when motifs of the USE were replaced by linker sequences. The addition of an oligoribonucleotide containing a USE motif to an in vitro polyadenylation reaction inhibits polyadenylation of a substrate representing the SV40 late polyadenylation signal, whereas a mutant oligoribonucleotide, a nonspecific oligoribonucleotide, and an oligoribonucleotide containing the Ul RNA-binding site had much reduced or no inhibitory effects. In addition, antibodies to bacterially produced, purified Ul snRNP-A protein specifically inhibit in vitro polyadenylation of the SV40 late substrate. These data suggest that the Ul snRNP-A protein performs an important role in polyadenylation through interaction with the USE. Because this interaction can occur when Ul snRNP-A protein is part of the Ul snRNP, our data provide evidence to support a link between the processes of splicing and polyadenylation, as suggested by the exon definition model.

We have previously shown that the U1 snRNP-A protein {U1A) interacts with elements in the SV40 late polyadenylation signal and that this association increases polyadenylation efficiency. It was postulated that this interaction occurs to facilitate protein-protein association between components of the U1 snRNP and proteins of the polyadenylation complex. We have now used GST fusion protein experiments, coimmunoprecipitations and Far Western blot analyses to demonstrate direct binding between U1A and the 160-kD subunit of cleavage--polyadenylation specificity factor (CPSF}. In addition, Western blot analyses of fractions from various stages of CPSF purification indicated that U1A copurified with CPSF to a point but could be separated in the highly purified fractions. These data suggest that UIA protein is not an integral component of CPSF but may be able to interact and affect its activity. In this regard, the addition of purified, recombinant U1A to polyadenylation reactions containing CPSF, polyIA) polymerase, and a precleaved RNA substrate resulted in concentration-dependent increases in both the level of polyadenylation and polylA} tail length. In agreement with the increase in polyadenylation efficiency caused by U1A, recombinant U1A stabilized the interaction of CPSF with the AAUAAA-containing substrate RNA in electrophoretic mobility shift experiments. These findings suggest that, in addition to its function in splicing, U1A plays a more global role in RNA processing through effects on polyadenylation.[ Moore et al. 1993; Sachs and Wahle 1993}. Splicing involves removal of intronic sequences and ligation of exons by a complex set of small nuclear ribonucleoprotein particles (snRNPsl and other factors known collectively as the spliceosome. Polyadenylation is the process by which the 3' end is formed through specific endonucleolytic cleavage of the precursor RNA and the addition of -250 adenosine residues. Both in vitro (Niwa et al. 1990;Berget 1991} and in vivo (Chiou et al. 1991;Nesic et al. 1993; Nesic and Maquat 1994} experiments 4These authors contributed equally to this work. SCorresponding author.have suggested that the processes of splicing and polyadenylation might be functionally linked. This proposal has been supported by our previous report that the U1 snRNP-A protein (U1A) interacts with elements in the SV40 late polyadenylation signal and that these interactions increase polyadenylation efficiency (Lutz and A1-wine 1994}. These data support the exon definition model of Berget and co-workers (for review, see Berget 1995}, which suggests that components of both the spliceosome and the polyadenylation complex may interact to define the last exon and affect the efficiencies of polyadenylation and last intron removal.There are currently five established mammalian factors comprising the complex that cleaves and polyadenylates substrate RNAs: cleavage-polyadenylation specificity factor (CPSF), cleavage stimulatory factor (CstF), polYIA) polymerase {PAP}, and cleavage factors I and II (CFI and CFII}...

Regulation of gene expression by RNA processing mechanisms is now understood to be an important level of control in mammalian cells. Regulation at the level of RNA transcription, splicing, polyadenylation, nucleo-cytoplasmic transport, and translation into polypeptides has been well-studied. Alternative RNA processing events, such as alternative splicing, also have been recognized as key contributors to the complexity of mammalian gene expression. Pre-messenger RNAs (pre-mRNAs) may be polyadenylated in several different ways due to more than one polyadenylation signal, allowing a single gene to encode multiple mRNA transcripts. However, alternative polyadenylation has only recently taken the field as a major player in gene regulation. This review summarizes what is currently known about alternative polyadenylation. It covers results from bioinformatics, as well as those from investigations of viral and tissue-specific studies and, importantly, will set the stage for what is yet to come.

Alternative RNA processing mechanisms, including alternative splicing and alternative polyadenylation, are increasingly recognized as important regulators of gene expression. This article will focus on what has recently been described about alternative polyadenylation in development, differentiation, and disease in higher eukaryotes. We will also describe how the evolving global methodologies for examining the cellular transcriptome, both experimental and bioinformatic, are revealing new details about the complex nature of alternative 3′ end formation, as well as interactions with other RNA-mediated and RNA processing mechanisms.

Cancer as we know it is actually an umbrella term for over 100 very unique malignancies in various tissues throughout the human body. Each type, and even subtype of cancer, has different genetic, epigenetic, and other cellular events responsible for malignant development and metastasis. Recent work has indicated that microRNAs (miRNAs) play a major role in these processes, sometimes by promoting cancer growth and other times by suppressing tumorigenesis. miRNAs are small, noncoding RNAs that negatively regulate expression of specific target genes. This review goes into an in‐depth look at the most recent finding regarding the significance of one particular miRNA, miR‐146a‐5p, and its involvement in cancer. Target gene validation and pathway analysis have provided mechanistic insight into this miRNA's purpose in assorted tissues. Additionally, this review outlines novel findings that suggest miR‐146a‐5p may be useful as a noninvasive biomarker and as a targeted therapeutic in several cancers. This article is categorized under: RNA in Disease and Development > RNA in Disease Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs