Long-distance regulation of Add2 pre-mRNA3′end processing

Abstract

Accurate 3′end processing depends on the correct recognition of polyadenylation regulatory elements by specific protein complexes. In addition to the well-known hexanucleotide motif and downstream sequence element (DSE), less-defined auxiliary elements are usually found to modulate cleavage and polyadenylation. They are generally located in close proximity to the core polyadenylation elements but, in most of the cases, the molecular mechanisms involved are not well defined. We concentrated our studies on the regulation of the mouse β adducin (Add2) pre-mRNA cleavage and polyadenylation. It contains two proximal erythroid-specific (PAS1 and PAS2-3) and one distal brain-specific (PAS4) polyadenylation sites along the 3′UTR. Using an in vivo approach based in the transfection of minigenes containing the Add2 polyadenylation signals, we previously identified the core regulatory elements responsible for PAS4 activity. Here, we have identified two novel non-canonical cis-acting elements regulating 3′end processing at PAS4, which show long-distance activities. The first of these elements, which spans for 257 nucleotides and is located at more than 5 kb upstream the PAS4, was essential to enable processing at the Add2 PAS4. The second element, located at about 4.5 kb upstream of the PAS4, reduces PAS4 processing. Both elements display long-distance activities and, to our knowledge, long-distance upstream polyadenylation regulatory elements have not been previously described in non-viral eukaryotic transcripts. These results highlight the complexity of the regulatory mechanisms directing Add2 pre-mRNA 3′end processing, and suggests that pre-mRNA 3′ end processing of other genes may also be unexpectedly regulated by non-canonical auxiliary elements.

TDP-43 regulatesβ-adducin(Add2) transcript stability

“…28 We utilized a 324 nt-long input RNA probe having 289 nt complementary to the pre-mRNA transcript (205 nt upstream and 84 nt downstream of the A4 PAS cleavage site) ( Since polyA tail length could be reduced in the absence of TDP-43, with an impact on mRNA stability, we performed the ligationmediated polyA length test (LM-PAT) assay to determine polyA tail length in the same siLUC and siTDP-treated RNA samples. As shown in Fig.…”

“…The radioactive RNA probe was prepared as described. 28 Approximately 500 cps of the probe were used for the hybridization reactions. pUC19 plasmid, digested with TaqI and NdeI, was labeled with (a¡ 32 P)-dCTP and used to estimate the size of the observed bands.…”

“…15 RNase protection assay (RPA) The RNase protection assays were performed as described previously. 28 Briefly, RNA samples were prepared from total RNA of transfected HeLa cells, treated with siRNA for Luciferase or TDP-43, as described above. Total RNA was treated with DNase, acid phenol-chloroform purified, ethanol precipitated and resuspended in R-loop buffer.…”

Cell Cycle Kinase Polo Is Controlled by a Widespread 3′ Untranslated Region Regulatory Sequence in Drosophila melanogaster

“…In mammals, this is in part achieved by the existence of auxiliary cis elements upstream or downstream of the pA signal in the pre-mRNA, such as upstream sequence elements (USEs) that influence cleavage and polyadenylation in an orientation-and position-dependent manner (32)(33)(34). USEs are typically pyrimidine-or U-rich sequences that were initially identified in viruses (35)(36)(37)(38)(39)(40) and subsequently described in human genes such as the C2 complement (41), COX-2 (42), MECP2 (43), F2 (44), collagen (45), and ADD1 (46) genes. However, USEs had not been identified in D. melanogaster yet.…”

A short 3’UTR motif regulates gene expression in bilaterians

“…Usage of non-canonical PAS is enhanced by the presence of auxiliary cis-elements in the pre-mRNA, such as the upstream sequence elements (USEs). USEs have been well characterized in mammals as pyrimidine or U-rich sequences that modulate cleavage and polyadenylation in an orientation-and position-dependent manner [11][12][13][14][15][16][17]. In humans, USEs modulate of PAS selection and 3' end formation efficiency by acting as an extra-platform for the recruitment of trans-acting factors, such as RBPs, with an important function in diseases, such as thrombosis [12,15,16,18,19].…”