Polyadenylation of ribosomal RNA in human cells

Abstract

The addition of poly(A)-tails to RNA is a process common to almost all organisms. In eukaryotes, stable poly(A)-tails, important for mRNA stability and translation initiation, are added to the 3′ ends of most nuclear-encoded mRNAs, but not to rRNAs. Contrarily, in prokaryotes and organelles, polyadenylation stimulates RNA degradation. Recently, polyadenylation of nuclear-encoded transcripts in yeast was reported to promote RNA degradation, demonstrating that polyadenylation can play a double-edged role for RNA of nuclear origin. Here we asked whether in human cells ribosomal RNA can undergo polyadenylation. Using both molecular and bioinformatic approaches, we detected non-abundant polyadenylated transcripts of the 18S and 28S rRNAs. Interestingly, many of the post-transcriptionally added tails were composed of heteropolymeric poly(A)-rich sequences containing the other nucleotides in addition to adenosine. These polyadenylated RNA fragments are most likely degradation intermediates, as primer extension (PE) analysis revealed the presence of distal fragmented molecules, some of which matched the polyadenylation sites of the proximal cleavage products revealed by oligo(dT) and circled RT–PCR. These results suggest the presence of a mechanism to degrade ribosomal RNAs in human cells, that possibly initiates with endonucleolytic cleavages and involves the addition of poly(A) or poly(A)-rich tails to truncated transcripts, similar to that which operates in prokaryotes and organelles.

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

“…A nuclear RNA surveillance mechanism involving polyadenylation and rapid degradation of nonfunctional, antisense, and noncoding RNAs has been described in yeast and human cells (4,9,(33)(34)(35)(36). We initially assumed that the truncated, polyadenylated rRNA molecules that we had previously detected in human cells (18) are solely related to this process, but in this study we found that transient adenylation can occur in the cytoplasm as well, as such molecules were detected in cytoplasmic fractions. However, in yeast, unstable transcripts derived from intergenic regions (i.e., nuclear) were reported to be stabilized upon inhibition of Xrn1 and Dcp1 (i.e., cytoplasmic).…”

“…In earlier work, we detected truncated 28S and 18S rRNAs containing homo-or heteropolymeric tails (18). It was plausible that these rRNA fragments are degradation intermediates of the nuclear process mentioned earlier, but a second option could not be ignored: transient, perhaps degradation-assisting, adenylation of RNA within the cytoplasm, coexisting with the stable poly(A) tails that characterize this cellular location.…”

“…Third, in any case, in previous work, we traced truncated adenylated 28S rRNA molecules to an endonucleolytically cleaved region of the 28S transcript (18). Multiple endonucleolytic cleavage events of rRNA or mRNA before transient tail addition, as in other poly(A)-mediated RNA decay pathways, could provide bare 5′ (and 3′) ends, thereby bypassing the need to decap the molecule (18). More studies are needed to characterize any differences between poly(A)-mediated rRNA and mRNA decay.…”

“…Previously, we isolated truncated, poly(A)-tailed ribosomal RNA molecules from total RNA of human cells (18). Interestingly these tails were either of homopolymeric [poly(A)] or heteropolymeric [poly(A)-rich] nature.…”

The PNPase, exosome and RNA helicases as the building components of evolutionarily-conserved RNA degradation machines

“…When PNPase or the archaeal exosome is the polymerizing enzyme, heteropolymeric tails composed mostly of adenosine but also of other nucleotides are produced [5,8,36,66]. Heteropolymeric tails were also recently identified in human cells [67].…”