Analysis of the \u3b2-adducin gene: new insights into gene structure and function

Adducins are a family of membrane skeleton proteins encoded by three related genes (ADD1, ADD2 and ADD3 or a-, b- and g-adducin genes). Both ADD1 and ADD3 are ubiquitously expressed, while the b-adducin gene shows a pattern of expression restricted to brain and haematopoietic tissues. Adducin is found as either a heterodimer or heterotetramer of a/b or a/g subunit composition in most tissues. Human erythrocytes mainly contain a/b heterodimers, whereas the g-subunit is also found at low levels in mouse red blood cells (RBCs). [...

Characterization of the distal polyadenylation site of the ß-adducin (Add2) pre-mRNA.

Most genes have multiple polyadenylation sites (PAS), which are often selected in a tissue-specific manner, altering protein products and affecting mRNA stability, subcellular localization and/or translability. Here we studied the polyadenylation mechanisms associated to the beta-adducin gene (Add2). We have previously shown that the Add2 gene has a very tight regulation of alternative polyadenylation, using proximal PAS in erythroid tissues, and a distal one in brain. Using chimeric minigenes and cell transfections we identified the core elements responsible for polyadenylation at the distal PAS. Deletion of either the hexanucleotide motif (Hm) or the downstream element (DSE) resulted in reduction of mature mRNA levels and activation of cryptic PAS, suggesting an important role for the DSE in polyadenylation of the distal Add2 PAS. Point mutation of the UG repeats present in the DSE, located immediately after the cleavage site, resulted in a reduction of processed mRNA and in the activation of the same cryptic site. RNA-EMSA showed that this region is active in forming RNA-protein complexes. Competition experiments showed that RNA lacking the DSE was not able to compete the RNA-protein complexes, supporting the hypothesis of an essential important role for the DSE. Next, using a RNA-pull down approach we identified some of the proteins bound to the DSE. Among these proteins we found PTB, TDP-43, FBP1 and FBP2, nucleolin, RNA helicase A and vigilin. All these proteins have a role in RNA metabolism, but only PTB has a reported function in polyadenylation. Additional experiments are needed to determine the precise functional role of these proteins in Add2 polyadenylation

TDP-43 regulates <i>β-adducin</i> (<i>Add2</i>) transcript stability

<div><p>TDP-43 is an RNA-binding protein involved in several steps of mRNA metabolism including transcription, splicing and stability. It is also involved in ALS and FTD, neurodegenerative diseases characterized by TDP-43 nuclear depletion. We previously identified TDP-43 as a binder of the downstream element (DSE) of the <i>β-Adducin</i> (<i>Add2</i>) brain-specific polyadenylation site (A4 PAS), suggesting its involvement in pre-mRNA 3′ end processing. Here, by using chimeric minigenes, we showed that TDP-43 depletion in HeLa and HEK293 cells resulted in down-regulation of both the chimeric and endogenous <i>Add2</i> transcripts. Despite having confirmed TDP-43-DSE in vitro interaction, we demonstrated that the in vivo effect was not mediated by the TDP-43-DSE interaction. In fact, substitution of the <i>Add2</i> DSE with viral <i>E-SV40</i> and <i>L-SV40</i> DSEs, which are not TDP-43 targets, still resulted in decreased <i>Add2</i> mRNA levels after TDP-43 downregulation. In addition, we failed to show interaction between TDP-43 and key polyadenylation factors, such as CstF-64 and CPSF160 and excluded TDP-43 involvement in pre-mRNA cleavage and regulation of polyA tail length. These evidences allowed us to exclude the pre-hypothesized role of TDP43 in modulating 3′ end processing of <i>Add2</i> pre-mRNA. Finally, we showed that TDP-43 regulates <i>Add2</i> gene expression levels by increasing <i>Add2</i> mRNA stability. Considering that <i>Add2</i> in brain participates in synapse assembly, synaptic plasticity and their stability, and its genetic inactivation in mice leads to LTP, LTD, learning and motor-coordination deficits, we hypothesize that a possible loss of Add2 function by TDP-43 depletion may contribute to ALS and FTD disease states.</p></div

Detection of brain- and spleen-specific β-adducin exons in rats and mice

<p><b>Copyright information:</b></p><p>Taken from "Brain-specific promoter and polyadenylation sites of the β-adducin pre-mRNA generate an unusually long 3′-UTR"</p><p>Nucleic Acids Research 2006;34(1):243-253.</p><p>Published online 9 Jan 2006</p><p>PMCID:PMC1326019.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> () Schematic representation of the riboprobes used in the RNase protection experiment shown in (B and C). The input probe and the size of the protected fragments for each probe and each tissue are indicated. ( and ) The spleen and brain probes shown in (A) were used in (B and C), respectively. Thirty micrograms of total spleen and brain RNA were annealed to the probe, digested with RNase, run in a polyacrylamide denaturing gel and autoradiographed. Lanes 1 and 7 correspond to the undigested probe (input probe), lanes 6 and 7 are radioactive molecular weight markers, lanes 2–3 and 9–10 spleen RNA, and lanes 4–5 and 11–12 brain RNAs. The arrows indicate the protected fragments. () Primer extension experiment with a primer annealing in the boundary between the constitutive 99 bp and the 217 bp exons (exons 2 and 3), that are present in both the brain and spleen forms of the β-adducin mRNA. The arrows indicate the primer extension products observed in brain and spleen RNAs. Lanes 15–16 and 17–18 correspond to spleen and brain RNAs, respectively. Lane 14 is a radioactive molecular weight marker, and lane 18 is a one-lane Sanger sequence using the same primer used in the experiment. () Northern blot experiment of cerebellum, brain and spleen mouse RNA (lanes 19–21, respectively) with a probe corresponding to the mouse brain-specific exon. The position of the 28S and 18S rRNA is indicated

Point mutations in the DSE also result in the activation of cryptic PAS.

<p><b>Panel A:</b> Scheme of the constructs used in Panel B. The DSE and USE were mutated (indicated by an asterisk) as shown in detail in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058879#s2" target="_blank">Materials and Methods</a> section. <b>Panel B:</b> Northern blot of RNA prepared from HeLa (lanes 1–4) and HEK293 (lanes 5–8) cells transfected with the constructs shown in Panel A, hybridized with the mß-Add 16ex (top panel) or the GFP probes (bottom panel). The position of the rRNAs is indicated, as well as the position of the expected RNA bands (indicated by brackets). The bar graph indicates the relative levels of expression normalized with the GFP signal (Add2/GFP ratio, mean±SD of three different experiments). <b>Panel C:</b> RACE-PCR experiment of the HeLa cells transfection to map the cleavage site of Add2 transcripts. Only the second semi-nested PCR reaction is shown. The Add2 polyadenylation sites are indicated: “A” and “D”, for the plasmid or brain mRNAs, “B” and “C” for the cryptic sites. Identical results were obtained in HEK293 cells.</p

The minigene-encoded transcript is cleaved and polyadenylated at the distal PAS.

<p><b>Panel A:</b> Scheme showing the homology among vertebrates in the last exon of the Add2 gene (UCSC browser). The A1, A23 and A4 PAS are indicated. The stop codon is indicated (“Stop”). <b>Panel B:</b> Scheme of the last exon of the mouse Add2 gene (top). The regions used for the generation of the A1-A23-A4 construct. The numbering refers to the first base of the last Add2 exon. The probe used in the Northern blots experiments is indicated. The expected size of the transcripts originating from the different PAS is shown. <b>Panel C:</b> Northern blot experiment of RNA prepared from HeLa cells transfected with the A1-A23-A4 construct. The position of the Add2 brain mRNA (lane 1), expected plasmid encoded transcripts (lane 2) and rRNA are indicated. Mouse brain RNA (mBR) was used as control.</p

An RNA lacking the DSE is not able to form RNA-protein complexes under more stringent incubation conditions.

<p><b>Panel A:</b> Scheme of the RNA probes and competitors used in Panel B. Panel B: RNA-EMSA experiment using complete HeLa nuclear extract and the RNA probes of Panel A. The different radioactive RNA probes were incubated with 2 µg of HeLa nuclear extract and 15 µg/µl of heparin to generate more stringent incubation conditions. The experiment was performed in more stringent conditions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058879#s2" target="_blank">Materials and Methods</a> section for details) than those used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058879#pone-0058879-g005" target="_blank">Figure 5</a> to make more evident the differences in complex formation efficiency.</p