Addition of poly(A) and poly(A)-rich tails during RNA degradation in the cytoplasm of human cells

Abstract

Polyadenylation of RNA is a posttranscriptional modification that can play two somewhat opposite roles: stable polyadenylation of RNA encoded in the nuclear genomes of eukaryote cells contributes to nuclear export, translation initiation, and possibly transcript longevity as well. Conversely, transient polyadenylation targets RNA molecules to rapid exonucleolytic degradation. The latter role has been shown to take place in prokaryotes and organelles, as well as the nucleus of eukaryotic cells. Here we present evidence of hetero-and homopolymeric adenylation of truncated RNA molecules within the cytoplasm of human cells. RNAi-mediated silencing of the major RNA decay machinery of the cell resulted in the accumulation of these polyadenylated RNA fragments, indicating that they are degradation intermediates. Together, these results suggest that a mechanism of RNA decay, involving transient polyadenylation, is present in the cytoplasm of human cells.exosome | heteropolymeric tails | RNA polyadenylation R NA polyadenylation occurs throughout the biological world.Stable poly(A) is associated with the mature 3′ end of most mRNAs encoded in the nuclear genome of eukaryotes. It is important for nuclear export and efficient translation initiation and may also contribute to transcript longevity (1). In turn, mRNA degradation in the cytoplasm usually initiates with deadenylation of the stable poly(A) tail, followed by either further degradation of the mRNA body by the exosome (3′-5′ pathway) or removal of the 5′ cap and subsequent 5′-3′ degradation by hXrn1 (exoribonuclease 1; 5′-3′ pathway) (2-4).Unlike stable poly(A), transient poly(A) is not associated with the mature 3′ end of the transcript. RNA decay pathways that use transient poly(A) include a stage in which poly(A) or poly (A)-rich tails are added to the 3′ ends of truncated degradation intermediates and are believed to assist exoribonucleases in their rapid degradation. As the tail addition can occur after endonucleolytic cleavage of the RNA or repetitive adenylation and 3′-5′ digestion, it often appears to be at "internal" positions relative to the full RNA sequence (5, 6). The term "transient" is used because the short-lived tail is degraded along with the RNA fragment. Transient poly(A) was first disclosed in Escherichia coli and then in additional bacteria, organelles, archaea and, eventually, in yeast and human nuclei (4,5,(7)(8)(9). In all systems in which truncated, polyadenylated RNA fragments were initially detected, they were later found to be correlated to poly(A)-assisted RNA decay (4,7,8).In yeast nuclei, transient poly(A) was found to play a role in RNA quality control wherein polyadenylation, initiated by the TRAMP complex, targets incorrectly folded tRNA molecules to degradation by the nuclear exosome (10, 11). In human cells, cotranscriptionally cleaved 3′ regions of an introduced β-globin gene and a class of short, highly unstable RNAs, dubbed PROMPTs (promoter upstream transcripts), were found to be adenylated and accumulated when the exosome wa...

Crystal structures of CRISPR-associated Csx3 reveal a manganese-dependent deadenylation exoribonuclease

“…In addition to functioning as the stabilizing element in eukaryotes, polyadenylation is also involved in the RNA degradation process throughout all life dom-ains. [32][33][34][35][36] During the decay process, RNA is sequentially cleaved by endonuclease, added with poly (A) or poly (A)-rich tail, and degraded by hydrolytic exoribonucleases. In archaea, exosomelike complexes are known to accomplish the process of RNA polyadenylation and exonucleolytic degradation.…”

The exozyme model: A continuum of functionally distinct complexes

“…For example, recombinant Sc Dis3 and TRAMP complexes were sufficient for the surveillance and turnover of hypomodified tRNA i Met in vitro, a function that was initially attributed to the entire exosome complex (Kadaba et al 2004(Kadaba et al , 2006LaCava et al 2005;Vanacova et al 2005;Schneider et al 2007). Human Dis3 has also been shown to have 28S rRNA processing functions that are distinct from three other exosome subunits (Slomovic et al 2010). Finally, Rrp46 homodimers have been shown to bind and degrade RNA in vitro (Yang et al 2010).…”

Ending the message: poly(A) signals then and now

“…Secondly, alternative poly(A) polymerases have been identified in most eukaryotes (Trf4 and Trf5 in Saccharomyces cerevisiae) that cooperate with the nuclear RNA degradation apparatus to promote degradation of unwanted or misprocessed transcripts (Schmid and Jensen 2008). RNA degradation-associated oligo(A) tailing Slomovic et al 2010) can similarly generate oligo(dT)-primed cDNA that is unrelated to authentic mRNA 39-terminal poly(A) tails. Finally, reverse transcriptase can, in some cases, ''misbehave'' by template switching from the original RNA template to the newly synthesized cDNA (Houseley and Tollervey 2010).…”