# Targeting the polyadenylation signal of Pre-mRNA: A new gene silencing approach for facioscapulohumeral dystrophy

Anne Charlotte Marsollier, Romain Joubert, Virginie Mariot, Julie Dumonceaux

► To cite this version:

Anne Charlotte Marsollier, Romain Joubert, Virginie Mariot, Julie Dumonceaux. Targeting the polyadenylation signal of Pre-mRNA: A new gene silencing approach for facioscapulohumeral dystrophy. International Journal of Molecular Sciences, 2018, 19 (5), 10.3390/ijms19051347. [https://doi.org/10.3390/ijms19051347](https://doi.org/10.3390/ijms19051347)

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Review

# Targeting the Polyadenylation Signal of Pre-mRNA: A New Gene Silencing Approach for Facioscapulohumeral Dystrophy

Anne-Charlotte Marsollier<sup>1,2</sup>, Romain Joubert<sup>1 ID</sup>, Virginie Mariot<sup>1 ID</sup> and Julie Dumonceaux<sup>1,*</sup>

Received: 14 March 2018; Accepted: 30 April 2018; Published: 3 May 2018

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**Abstract:** Facioscapulohumeral dystrophy (FSHD) is characterized by the contraction of the D4Z4 array located in the sub-telomeric region of the chromosome 4, leading to the aberrant expression of the DUX4 transcription factor and the mis-regulation of hundreds of genes. Several therapeutic strategies have been proposed among which the possibility to target the polyadenylation signal to silence the causative gene of the disease. Indeed, defects in mRNA polyadenylation leads to an alteration of the transcription termination, a disruption of mRNA transport from the nucleus to the cytoplasm decreasing the mRNA stability and translation efficiency. This review discusses the polyadenylation mechanisms, why alternative polyadenylation impacts gene expression, and how targeting polyadenylation signal may be a potential therapeutic approach for FSHD.

**Keywords:** polyadenylation; alternative polyadenylation; muscular diseases; facioscapulohumeral dystrophy; myotonic dystrophy; therapeutic strategies

## 1. Introduction

Several molecules targeting RNAs have been developed during the last few years to treat patients affected by muscle diseases, including RNA interference and synthetic antisense oligonucleotides (AON). These molecules can (i) prevent formation of the mRNA 5'cap; (ii) modulate RNA splicing by masking key sequences; (iii) influence the use of an alternative polyadenylation site; (iv) induce a RNAse H1-dependent degradation of the mRNA, and (v) inhibit the mRNA translation via steric blockade of ribosome access to mRNA (for review see [1]). In this review, another possibility is investigated: targeting the polyadenylation signals to destabilize the pre-mRNA.

## 2. Polyadenylation Mechanisms

The vast majority of eukaryotic mRNAs, with the exception of replication dependent histone mRNAs, present a similar 3'end characterized by a long chain of adenine nucleotides called the poly(A) tail. This poly(A) tail, which is not encoded by the DNA, is formed after endonucleolytic cleavage of the primary transcript followed by the addition of adenine nucleotides at the 3'OH end of the cleavage product. The specificity and efficiency of this 3'end processing are determined by the binding of a multiprotein complex to regulatory cis-acting sequence elements on the pre-mRNA.

<footnote n="1"><sup>1</sup> NIHR Biomedical Research Centre, University College London, Great Ormond Street Institute of Child Health and Great Ormond Street Hospital NHS Trust, London WC1N 1EH, UK; annecharlotte.marsollier@gmail.com (A.-C.M.); r.joubert@ucl.ac.uk (R.J.); virginie.mariot@ucl.ac.uk (V.M.)</footnote>

<footnote n="2"><sup>2</sup> Laboratoire Reproduction et Développement des plantes, Université de Lyon, ENS de Lyon, UCB Lyon 1, CNRS, INRA, F-69342 Lyon, France</footnote>

<footnote>* Correspondence: j.dumonceaux@ucl.ac.uk; Tel.: +44-(0)2-0790-52860</footnote>
## 2.1. Cis-Regulatory Sequence Elements

Few regulatory sequences on the pre-mRNA play a major role in the 3'end processing. The most important sequence element is probably the polyadenylation (poly(A)) signal (PAS), defined by an hexameric consensus sequence (usually A[A/U]UAAA) located ~20-30 nucleotides upstream of the cleavage site (Figure 1A). This hexamer is not strictly conserved, and several variants have been identified [2-4] (Table 1). These variants are not as efficient as the canonical one and the analysis of their distribution suggests they may be used for regulatory purposes (Table 1) [2-5]. Polymorphisms in PAS are rare, highlighting the importance of the sequence conservation in evolution [6]: alterations in the PAS have been frequently associated with diseases, such as β-thalassemia (OMIM #613985) or immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX, OMIM #304790) (For review see [7]).

The second regulatory sequence is the downstream sequence element (DSE), characterized by a high density of uracil (U) and/or guanine/uracil (G/U) residues and located more than 30 nucleotides downstream of the cleavage site [8-10] (Figure 1). There is no clear consensus sequence for the DSE, but its nucleotide composition seems to impact the 3'end processing efficiency [11,12].

A third cis-regulatory sequence is the cleavage site, located between 20 and 30 nucleotides downstream of the PAS (Figure 1) [4,13]. The poly(A) tail attachment begins at this cleavage site (frequently at a CA or a UA dinucleotide in mammals). The nucleotide composition around the cleavage site is heterogeneous and importantly, impacts the cleavage efficiency [14,15].

Finally, additional auxiliary sequences, located upstream or downstream of the cleavage site, can also influence the 3'end processing efficiency: (i) U-rich auxiliary sequences (USE) [3,9]; (ii) G-rich auxiliary sequence elements leading to the formation of G-quadruplex structures [9,16,17]; (iii) AUA auxiliary element for a selected set of mRNAs polyadenylated by the non-canonical poly(A) polymerase Star-PAP [15,18-21]; or (iv) distal auxiliary elements located downstream from the PAS [22].

<table><caption>**Table 1.** Polyadenylation signals frequencies in humans and mice.</caption><thead><tr><th>Hexameric Sequences</th><th>Human [2]</th><th>Human [4]</th><th>Mouse [4]</th></tr></thead><tbody><tr><td>AAUAAA</td><td>58.2</td><td>53.18</td><td>59.16</td></tr><tr><td>AUUAAA</td><td>14.9</td><td>16.78</td><td>16.11</td></tr><tr><td>UAUAAA</td><td>3.2</td><td>4.37</td><td>3.79</td></tr><tr><td>AGUAAA</td><td>2.7</td><td>3.72</td><td>3.28</td></tr><tr><td>AAGAAA</td><td>1.1</td><td>2.99</td><td>2.15</td></tr><tr><td>AAUAUA</td><td>1.7</td><td>2.13</td><td>1.71</td></tr><tr><td>AAUACA</td><td>1.2</td><td>2.03</td><td>1.65</td></tr><tr><td>CAUAAA</td><td>1.3</td><td>1.92</td><td>1.80</td></tr><tr><td>GAUAAA</td><td>1.3</td><td>1.75</td><td>1.16</td></tr><tr><td>AAUGAA</td><td>0.8</td><td>1.56</td><td>0.90</td></tr><tr><td>UUUAAA</td><td>1.2</td><td>1.20</td><td>1.08</td></tr><tr><td>ACUAAA</td><td>0.6</td><td>0.93</td><td>0.64</td></tr><tr><td>AAUAGA</td><td>0.7</td><td>0.60</td><td>0.36</td></tr><tr><td>AAAAAG</td><td>0.8</td><td>-</td><td>-</td></tr><tr><td>AAAACA</td><td>0.5</td><td>-</td><td>-</td></tr><tr><td>GGGGCU</td><td>0.3</td><td>-</td><td>-</td></tr></tbody></table>
<figure><img src="image_4.png" /><figcaption>**Figure 1.** Cis-regulatory sequence elements and protein factors involved in cleavage and polyadenylation. (A). The specificity and efficiency of 3'end processing is determined by the binding of more than 80 RNA-binding proteins to regulatory cis-acting RNA sequence elements including: the polyadenylation signal (PAS) A[A/U]UAAA; the cleavage site (represented by NN) and the downstream sequence element (DSE). Auxiliary sequences can be found near the polyadenylation signal or the DSE. The core processing complex, which is sufficient for the cleavage and polyadenylation, is composed of approximately 20 proteins, distributed in 8 complexes: the cleavage and polyadenylation specificity factor (CPSF), the cleavage stimulation factor (CstF); the mammalian cleavage factors I (CFIm) and the mammalian cleavage factors II (CFIIm); the single protein poly(A) polymerase (PAP); the single protein poly(A)-binding protein nuclear 1 (PABPN1); the single protein RNA polymerase II large subunit (Pol II); and the symplekin. Subunits of the different factors are indicated. (B). CPSF and CstF are co-transcriptionally recruited to the poly(A) signal and the DSE respectively, causing an endonucleolytic cleavage of the pre-mRNA between the PAS and the DSE at the cleavage site. Two fragments are generated: one fragment with a free 5'phosphate group which is rapidly degraded by exoribonucleases and one fragment with a free 3'hydroxyl group on which 250 adenines will be added by PAP. The newly synthetized poly(A) tail is covered by PAPBN1, allowing mRNA circularization and stabilization.</figcaption></figure>
## 2.2. Core Processing Complex

The cleavage and polyadenylation reactions are governed by more than 80 RNA-binding proteins but less than 20 factors compose the core of the processing complex and are necessary and sufficient to mediate cleavage and polyadenylation in vitro [23,24]. These 20 factors are distributed in eight complexes (Figure 1) [23,25]:

- **Cleavage and polyadenylation specificity factor (CPSF):** is a multiprotein complex implicated in the PAS recognition and the cleavage of the pre-mRNA [26–28]. The core of CPSF complex is composed of CPSF100 and CPSF73 which form a heterodimer and recruit the other CPSF subunits and symplekin [29,30]. CPSF73 has a zinc-dependent endonuclease activity that is essential for the pre-mRNA cleavage. It has a very weak enzymatic activity suggesting that other factors may be required for an efficient cleavage [31,32]. The specific interaction of CPSF with the hexameric poly(A) signal is mediated by WDR33, CPSF30, and CPSF160, while hFip1 binds the U-rich sequences [26,33–36]. Finally, hFip1 and CPSF160 recruit by direct interaction the poly(A) polymerase (PAP) to the PAS [26,37].

- **Cleavage stimulation factor (CstF):** is essential for the cleavage reaction but not for the polyadenylation reaction [38,39]. CstF is an multimeric protein complex made up of subunits CstF64, CstF77, and CstF50 [28,40] which are respectively involved in (i) the specific recognition of the DSE region (by CstF64), (ii) the assembly of the CstF complex (by CstF77), (iii) the CstF-CPSF interaction (strong interaction between CPSF160 and CstF77), (iv) the interaction with the C-terminal domain of the RNA polymerase PolII (PolII) (by CstF50), and (v) the interaction with the breast cancer 1 (BRCA1) associated really interesting new gene (RING) domain 1 (BARD1) complex (by CstF50) to inhibit the pre-mRNA 3'end processing during DNA repair/following DNA damage, reducing errors in the mRNA [41–44].

- **Symplekin:** is considered to be a scaffolding protein connecting CPSF and CstF and supporting the assembly of the polyadenylation machinery [27,28,45].

- **Mammalian cleavage factor I (CFIm):** influences alternative poly(A) site selection, mRNA transport and mRNA splicing [45–50]. It is a heterodimer composed of the smallest CFIm25 subunits and any of the largest CFIm68, CFIm59, or CFIm72 subunits [51–54]. CFIm binds UGUA motifs, typically located upstream of the PAS [9,55,56].

- **Mammalian cleavage factor II (CFIIm):** is required only for the cleavage step [57–59] and is composed of two subunits: CFIIAm, which is required for the cleavage reaction; and CFIIBm, which acts as a stimulator for the cleavage [23].

- **Poly(A) polymerase (PAP):** catalyzes the reaction leading to the addition of 200–250 adenosines as polyadenosine tail to the newly synthesized pre-mRNA molecules [60–62]. PAP is recruited by CPSF [26] and its activity is stimulated by the poly(A) binding protein nuclear 1 (PABPN1), which plays a major role in poly(A) tail length control [63–65]: the binding of PABPN1 to the newly synthesized polyadenosine tail accelerates the rate of adenosine addition mediated by PAP [62–64]. PABPN1 covers the entire length of the poly(A) tail during and after the polyadenylation reaction. When the poly(A) tail reaches 200–250 adenosines, the polyadenylation reaction is stopped [63]. The length of the poly(A) tail appears to be critical for a suitable gene expression: transcripts with short or long poly(A) tails are retained in the nucleus and degraded [66–68].

The RNA polymerase II (PolII) plays a critical role by coupling pre-mRNA processing to transcription [69,70]. The PolII C-Terminal Domain (CTD) may promote the assembly of a 3'-end processing complex through an interaction with CPSF, CstF, and CFIIm [71–73]. PolII may also be required for efficient pre-mRNA cleavage [71].

Besides these factors, other proteins participating in the 5' end capping can also influence the cleavage and polyadenylation efficiency [74–76].
## 2.3. Polyadenylation Steps

The first step of the 3'end processing is the co-transcriptional recruitment of CPSF and CstF to the PAS and the DSE respectively [77,78]. CPSF and CstF are loaded onto PolII during elongation [69,70,79] and after the transcription of the PAS, PolII pauses allowing the binding of CPSF to the hexameric sequence through WDR33 and CPSF30 [70]. CstF then interacts with the U/GU rich sequences in the DSE via CstF64 [80] and once the CPSF and CstF components are linked, additional factors—including CFIm and CFIIIm—join the complex around the future cleavage region [77]. The assembly of all of these proteins induces an endonucleolytic cleavage of the pre-mRNA between the PAS and the DSE, generally 20–30 nucleotide downstream of the hexameric sequence (Figure 1B). The cleavage efficiency can be influenced by 5' cap structure such as the nuclear cap-binding complex [75]. Two fragments are generated: one with a free 3'hydroxyl group and the other one with a free 5'phosphate group which is immediately degraded by the Xrn2 exoribonuclease (Figure 1) [81,82]. In parallel, PAP is anchored to the cleaved pre-mRNA by an interaction with CPSF and starts adding adenines [26,37]. The newly synthetized poly(A) tail is covered by PABPN1 proteins which control its length [62–65]. When the polyadenylation process is complete, the 3'end processing complex is disassembled. PABPN1 thus interacts with the initiation factor proteins located in the 5' untranslated region (UTR) leading to the mRNA circularization and stabilization [58,59,77,83,84]. The mature mRNA is then ready to be exported to the cytoplasm.

# 3. Alternative Polyadenylation

## 3.1. General Regulation of Alternative Polyadenylations

In mammals, 70–80% of transcripts have at least two alternative PAS leading to cleavage at different sites and production of different mRNA transcripts—a phenomenon called alternative polyadenylation (APA) [85,86]. A multitude of factors is involved in the selection of the 3'-end processing site. We will only focus on the most important and those described in muscle cells. Among these factors is the PAS hexameric sequence itself, which is not identical in the distal and proximal PAS sites. Most distal sites tend to use the canonical A[A/U]UAAA sequence whereas the proximal sites tend to use variant signals [2]. Most 3'-APA also tends to be associated with stronger and more conserved cis-regulatory sequence elements (upstream U-rich elements and DSE) [42]. Since the variant PAS signals are processed less efficiently than the canonical ones, the use of a proximal PAS may be done for regulatory purposes. However, it is still far from clear how a particular PAS is chosen and many factors may influence this choice. Alternative polyadenylation has been described to be regulated by many factors, including the cell proliferation and differentiation state, the distance between two PAS, the PolII polymerase speed, the presence of a pause site downstream of the proximal PAS, the presence of methylated CpG islands, nucleosome occupancy or histone methylation, and the concentration of the different proteins involved in the polyadenylation steps [2,87–99]. Splicing and polyadenylation are also interconnected and are likely to be in competition with each other in case of an intronic PAS. For example, ablation of the splicing factor 3B subunit1 (a component of U2 snRNP) activates intronic PAS [97]. U1 snRNP also affects cleavage and polyadenylation independently of its role in splicing. The use of cryptic PAS within introns close to the 5'-end of the transcript is increased in the case of knockdown of U1 snRNP, potentially because U1 snRNP binding to these regions blocks their recognition by cleavage factors [100,101].

## 3.2. Alternative Polyadenylations and Their Consequences

Several patterns define the APAs. The most frequent is the presence of multiple PAS in the 3'UTR of the terminal exon. Cleavage and polyadenylation at any of these sites will lead to transcript isoforms that differ in the length of the 3'UTR, with an identical protein-coding region (Figure 2A,B). Therefore, 3'UTR APA is more likely to affect post-transcriptional gene regulation through the modulation of mRNA stability, translation, nuclear export and cellular localization [86]. Although two recent articles
have demonstrated that 3′UTR shortening may have a limited effect on mRNA stability [102,103], isoforms with long 3′UTRs are believed to be less stable than short isoforms because they can bind more destabilizing elements. Global analyses have revealed that APA influences around 10% of predicted targets between any two cell types analyzed [104]. The length of the 3′UTR also influences the mRNA subcellular localization and long 3′UTR isoforms are also more abundant in the nucleus [105].

<figure><img src="image_5.png" /><figcaption>**Figure 2.** Alternative polyadenylations (APA). APAs have several patterns. The use of (A) a constitutive polyadenylation at a distal site leads to the normal mRNA and protein. (B) A proximal site located in a non-coding sequence of the mRNA results in 3′UTR shortening without any modification of the protein. (C) An APA located in a proximal exon (or in the coding sequence of the last exon) leads to truncated proteins. (D) An APA located in an intron (i.e., involving a cryptic poly(A) site in introns) and leads to a modified protein with an alternative C-terminus or to truncated proteins (depending of the presence of a stop codon). (E) An APA located in an alternative terminal exon APA, due to the use of an alternative splicing, leads to a protein with a different C-terminus. Introns are in grey or represented by a dotted line when spliced; non-coding sequence are hatched. Distal PAS is represented by a black arrowhead and proximal alternative PASs by a white one.</figcaption></figure>

Another pattern of APA is defined by the presence of a cryptic PAS in internal exons or in introns (Figure 2C,D). Transcripts produced from an internal exon or intronic APA cannot possess an in-frame stop codon or 3′UTR and are likely to be degraded rapidly through the non-stop mediated mRNA decay [106]. In the absence of a stop codon, the poly(A) tail could also generate a C-terminal poly-lysine tag which is generally unstable [107,108]. When present in the coding sequence, APAs can lead to the expression of truncated proteins with new functions as seen in the retinoblastoma binding protein 6 (OMIM #600938) [109–111].

A last form of APA, involving alternative exons, leads to proteins lacking domains or with alternative sequences (Figure 2E). For example, the immunoglobulin M (IgM) heavy chain has two transcripts, resulting in the shift from a distal PAS to an internal site and then the substitution of the two terminal exons, coding the amino acid sequence for membrane-binding, by a sequence involved in the secretion [112,113].

### 3.3. *Alternative Polyadenylations in the Skeletal Muscle World*

A growing number of studies about APAs has been published during recent years, in which APAs are associated with knockdown, depletion, or overexpression of different proteins or genes. Here, we will focus only on APAs connected to the “skeletal muscle world”.
APAs have been described to play a role in muscle metabolism and myogenesis. For example, (i) slow and fast muscle fibres display 7% of their transcriptome with different APAs [114]. (ii) *PAX3* and *PAX7*, coding for two transcription factors involved in myogenesis, are also subject to APAs resulting in resistance to miR-206 regulation or alternative C-terminal domain [115,116]. (iii) During myogenesis, differential expression, and localization of copper transporters are associated with mRNA 3′UTR shortening of the transporter *ATP7A* [117]. (iv) *UCP3* has an intronic APA leading to a protein without an inhibition site at the C-terminus [118]. This may play a role in the pathogenesis of dystrophies because UCP3 is involved in the mitochondrial proton leak and the limitation of reactive oxygen species production [119], and consequently in oxidative stress regulation. (v) One of the most recognized major players in controlling muscle mass is mammalian target of rapamycin (mTOR) whose activation increases protein synthesis and prevents atrophy (for review see [120]). One of the molecular signatures of mTOR activation includes 3′UTR shortening of mRNAs leading to the overexpression of selected E2 and E3 components in ubiquitin ligase complexes resulting in elevated levels of protein ubiquitination [121]. This phenomenon could be required for the continuous supply of amino acids to cellular systems, to maintain the steady-state protein synthesis [122]. (vi) Like mTOR, the androgen receptor (AR) is a well-known regulator of muscle anabolism (for review see [123]). Some prostate cancers are castration-resistant due to *AR* splice variants that are constitutively active transcription factors. These variants lack the ligand-binding domain thanks to the use of an APA in a cryptic exon [124].

PAS can also be blocked by protein and/or RNA elements that compete with the 3'-end processing machinery. For example, mutations in PABPN1 causes oculopharyngeal muscular dystrophy (OPMD, OMIM #164300), characterized by progressive degeneration of muscles in adults [125]. Mutated PABPN1 aggregates in the nucleus and forms filamentous nuclear inclusions. While this protein is not involved in the choice of PAS, its knockdown produced a shorter 3′UTR [126,127], suggesting that PABPN1 could act prior to cleavage and polyadenylation of pre-mRNA to determine the PAS used. It was proposed that PABPN1 competes with CPSF for binding to A-rich regions at proximal, consensual PAS inhibiting its usage. When PABPN1 is depleted or mutated (in OMPD), CPSF can recognize the previously hidden PAS. Interestingly, whereas PABPN1 is ubiquitously expressed and presumably contributes to control of gene expression in all tissues, mutation of the PABPN1 gene only affects a limited set of skeletal muscles, most likely because PABPN1 levels are dramatically lowered in skeletal muscle compared to other tissues [128], thus highlighting the importance of the concentration of the different proteins involved in the polyadenylation steps in APA.

APA patterns may also be involved in the onset of myotonic dystrophy (DM1, OMIM #160900). Indeed, DM is characterized by the re-emergence of developmentally immature alternative splicings (AS) and APA patterns in adult tissues because proteins of the MBLN family are titrated, leading to immature AS. Gene ontology and systems analysis reveals several different classes of misregulated genes in APA, including those involved in ubiquitination, IGF-1 signalling, and the mTOR pathway [129].

## 4. Therapeutic Strategies Targeting Polyadenylation in Muscle Diseases

Because polyadenylation is essential for gene expression, strategies aiming at disrupting gene expression by targeting the polyadenylation have been developed. These strategies might be particularly important for gain of function diseases such as DM1 or facioscaplohumaral dystrophy (FSHD, OMIM #158900) which are two of the three most prevalent muscle diseases with an estimated prevalence of 4/100,000 and 4.5/100,000, respectively.

DM1 is a multisystemic disease and patients show an extremely widely variable phenotype. The symptoms include myotonia, muscle wasting, cardiac conduction defects, cataracts, and insulin resistance (for review see [130]). DM1 is an inherited monogenic disorder characterized by a repeat expansion in the *Dystrophia Myotonica Protein Kinase* (DMPK) gene localized on chromosome 19. The 3′UTR of this gene normally contains 5–37 copies of a CTG trinucleotide repeat while the most
severely affected DM1 patients harbor between 50 and several thousand repeats [131]. The mutation is thought to adopt a stem-loop structure within the mRNA [132] and numerous mechanisms have been proposed to explain how CUG-expansion in the 3'UTR untranslated region of an mRNA creates such adverse multisystemic effects including the aberrant alternative splicing of several key mRNAs, the alterations in the usage of alternative polyadenylation sites of a number of mRNAs and diffusion of the molecular pathological phenotype through nuclear protein spreading (for review see [133]) [134] (Figure 3A). DM1 thus appears to be the result of a highly stable hairpin mRNA structure in the *DPMK* mRNA which facilitates binding/sequestration of several factors, mainly leading to the misregulation of several splicing events and dysregulation of translation.

<figure><img src="image_6.png" /><figcaption>**Figure 3.** Therapeutic strategies targeting polyadenylation in muscle diseases. (A) In Myotonic dystrophic type 1 patients, the mutated DPMK gene carries between 50 to 2500 CTG expansions in the 3'UTR leading to the formation of a stem-loop which sequesters splicing factors such as MBLN1, causing foci formation and splicing defects. (B) Two PAS (SV40p(A) and bGH p(A)) have been inserted between exons 9 and 10 (by the TALEN system), allowing the elimination of the mutant transcript. The presence of a stop codon located at the beginning of intron 9 leads to a truncated mRNA *DPMK* (Figure 2D) which no longer carries the toxic CUG repeats [135]. (C) *DUX4* ORF is located in each D4Z4 repeat but the polyadenylation signal is in exon 3 (E3) positioned in the sub-telomeric part of the chromosome 4. The hypomethylation of the D4Z4 region, when associated with a permissive chromosome 4, leads to the aberrant expression of the DUX4 transcription factor and the mis-regulation of hundreds of DUX4 target genes. (D) In the presence of the PMO-PAS targeting the DUX4 PAS, correct polyadenylation of DUX4 is inhibited, leading to an unstable *DUX4* mRNA which is not translated [136,137].</figcaption></figure>

FSHD pathology is characterized by an atrophy of the muscles of the face, shoulders, and arms, leading to muscle weakness and asymmetric involvement of affected musculature [138,139]. In 95% of FSHD patients (named FSHD1, OMIM #158900), a contraction of the D4Z4 array, located in the sub-telomeric region of chromosome 4, is observed [140]. This contraction is associated with a loss of repressive epigenetic marks within the D4Z4 macrosatellite, leading to the expression of the *DUX4* gene which is composed of three exons. The DUX4 ORF is fully included in the first exon, whereas exons 2 and 3 are non-coding regions (3'UTR). Importantly, exon 3 is located outside of the D4Z4 repeats and carries the *DUX4* PAS (Figure 3C) [141,142]. Two allelic variants (4qA and 4qB) exist in
this region distal to D4Z4 but FSHD is only associated with the 4qA variant [143] which contains a
functional but non-canonical PAS (AUUAAA) [141]. The remaining 5% of FSHD patients (named
FSHD2, OMIM #158901) do not show the D4Z4 contraction but carry a mutation in the epigenetic
modifier genes *SMCHD1* or *DNMT3B* [144,145], also leading to the hypomethylation of the D4Z4 array
and to the aberrant expression of the DUX4 protein in the context of a permissive chromosome 4. Even
if other genes may participate in the onset of FSHD [146,147] DUX4 is believed to play a major role in
disease onset and/or progression. Indeed, (i) DUX4 protein and mRNA are detected in both adult and
fetal FSHD1 and FSHD2 muscle cells and biopsies [148–150], (ii) hypomethylation of the D4Z4 array is
always observed in FSHD patients, (iii) individuals carrying a permissive chromosome 4 but lacking
the hypomethylation of the D4Z4 array are asymptomatic carriers [151], and (iv) the expression of
DUX4 accounts for the majority of the gene expression changes in FSHD skeletal muscles [152].

Both FSHD and DM1 are gain-of-function diseases and so far, there is no curative or preventive
treatment for these pathologies. Several strategies have been proposed in the literature, and in this
review we will focus on those altering the polyadenylation signal. Mutated *DMPK* and *DUX4*, being the
causative genes of DM1 and FSHD respectively, were targeted by strategies using either a TALEN-based
or an antisense oligonucleotide approach [135-137] (Figure 3B,D). In myotonic dystrophy, the authors
used an approach that they had previously developed on neural stem cells, which was the insertion of
two poly(A) signals upstream of *DMPK* CTG repeats in intron 9, thus leading to premature cleavage
of transcript before the transcription of the toxic region [153]. In this case, the introduced PASs may
have created an APA which inhibits intron 9 splicing and an in frame stop codon located at the
beginning of intron 9 is used (Figure 2D). The authors used DM1 induced pluripotent stem (iPS) cells
and demonstrated that integration of a PAS upstream of the CTG repeats eliminated nuclear RNA
foci in the treated cells, even after their differentiation into neural cells or cardiomyocytes. Aberrant
splicings were also abolished. Interestingly, when the PAS was inserted between the *DMPK* stop codon
and the start of the CTG repeats, none of the clones had the mutant allele targeted, potentially because
of heterochromatin spreading caused by the expanded CTG repeats.

In FSHD, a systematic analysis of the cis-acting elements that govern *DUX4* cleavage and
polyadenylation has been performed and revealed that sequences downstream of the SNP located
within the β-satellite region are critical for *DUX4* cleavage and polyadenylation [22]. Antisense
oligonucleotides targeting the mRNA of a GFP reporter construct carrying these distal auxiliary
elements led to a decrease in GFP expression, thus suggesting that these elements could have
therapeutic potential [22].

Two independent groups have targeted the *DUX4* PAS using antisense phosphorodiamidate
morpholino oligonucleotides (PMO) in FSHD cells [136,137]. Different 3'key elements of the *DUX4*
mRNA were targeted by several PMOs and the same PMO (PMO-PAS) was found to be the one giving
the best extinction of *DUX4* mRNA in both studies (Figure 3D). This PMO precisely targets the PAS
which is in a region of an open conformation, whereas the cleavage site and the DSE lay within a
closed region. In FSHD myotubes, *DUX4* downregulation in the presence of PMO-PAS is associated
with a downregulation of many transcriptional targets of DUX4 without particular off-target effects.
In vivo, after electroporation of PMOs into FSHD patient muscle xenografts in immunodeficient mice,
DUX4 target genes are also downregulated [136]. Remarkably, whereas other PAS are present in the
subtelomeric region of chromosome 4, downstream from the pathological one, none of them seems
to be used in the presence of the PMO [137]. However, one of the PMOs used in the Marsollier et
al. publication (PMO-CS3) induces a switch in cleavage site usage ~40 nt upstream of the normal
one [137], thus suggesting that an unidentified alternative PAS was used. The use of this APA allows
*DUX4* mRNA to escape, at least partially, the degradation process which occurs in non-polyadenylated
mRNA. Moreover, because this new cleavage site is located upstream of the normal one, it is possible
the truncated *DUX4* mRNA may be more stable. The mechanisms leading to the use of an upstream
APA are not known and need to be deciphered.
**5. Conclusions**

During the past 20 years, many laboratories have used antisense oligonucleotides to silence gene expression. Targeting the 3' end element of mRNA is a new approach that offers several advantages: (i) all polyadenylated mRNAs may be targeted using this strategy since polyadenylation is a crucial and common step required for the maturation of all eukaryote mRNAs (with the exception of replication dependent histone mRNA); (ii) genes with only one exon can be targeted whereas they are not eligible for other strategies such as exon skipping; (iii) in diseases characterized by the utilization of inadequate APAs, these APAs can be targeted to promote the use of the canonical PAS. This strategy thus presents an important clinical therapeutic potential not only for muscle diseases, but also for other genetic diseases such as cancer.

**Author Contributions:** All the authors wrote the paper.

**Acknowledgments:** All research at Great Ormond Street Hospital NHS Foundation Trust and UCL Great Ormond Street Institute of Child Health is made possible by the NIHR Great Ormond Street Hospital Biomedical Research Center. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. We thank Jennifer Morgan for careful reading of the article and editing the English. This work was funded by the FSH Society research grant FSHS-82015-06.

**Conflicts of Interest:** The authors declare no conflict of interest.

**References**

1. DeVos, S.L.; Miller, T.M. Antisense oligonucleotides: Treating neurodegeneration at the level of RNA. *Neurotherapeutics* **2013**, *10*, 486-497. [CrossRef] [PubMed]

2. Beaudoing, E.; Freier, S.; Wyatt, J.R.; Claverie, J.M.; Gautheret, D. Patterns of variant poly(A)denylation signal usage in human genes. *Genome Res.* **2000**, *10*, 1001-1010. [CrossRef] [PubMed]

3. Gruber, A.J.; Schmidt, R.; Gruber, A.R.; Martin, G.; Ghosh, S.; Belmadani, M.; Keller, W.; Zavolan, M. A comprehensive analysis of 3' end sequencing data sets reveals novel poly(A)denylation signals and the repressive role of heterogeneous ribonucleoprotein c on cleavage and poly(A)denylation. *Genome Res.* **2016**, *26*, 1145-1159. [CrossRef] [PubMed]

4. Tian, B.; Hu, J.; Zhang, H.; Lutz, C.S. A large-scale analysis of mRNA poly(A)denylation of human and mouse genes. *Nucleic Acids Res.* **2005**, *33*, 201-212. [CrossRef] [PubMed]

5. Proudfoot, N.J.; Brownlee, G.G. 3' non-coding region sequences in eukaryotic messenger rna. *Nature* **1976**, *263*, 211-214. [CrossRef] [PubMed]

6. Ferreira, P.G.; Oti, M.; Barann, M.; Wieland, T.; Ezquina, S.; FriedlÄnder, M.R.; Rivas, M.A.; Esteve-Codina, A.; Consortium, T.G.; Estivill, X.; et al. Sequence variation between 462 human individuals fine-tunes functional sites of RNA processing. *Sci. Rep.* **2016**, *6*, 32406. [CrossRef] [PubMed]

7. Curinha, A.; Oliveira Braz, S.; Pereira-Castro, I.; Cruz, A.; Moreira, A. Implications of poly(A)denylation in health and disease. *Nucleus* **2014**, *5*, 508-519. [CrossRef] [PubMed]

8. Gil, A.; Proudfoot, N.J. Position-dependent sequence elements downstream of AAUAAA are required for efficient rabbit beta-globin mRNA 3' end formation. *Cell* **1987**, *49*, 399-406. [CrossRef]

9. Hu, J.; Lutz, C.S.; Wilusz, J.; Tian, B. Bioinformatic identification of candidate cis-regulatory elements involved in human mRNA poly(A)denylation. *RNA* **2005**, *11*, 1485-1493. [CrossRef] [PubMed]

10. Zarudnaya, M.I.; Kolomiets, I.M.; Potyahaylo, A.L.; Hovorun, D.M. Downstream elements of mammalian pre-mRNA poly(A)denylation signals: Primary, secondary and higher-order structures. *Nucleic Acids Res.* **2003**, *31*, 1375-1386. [CrossRef] [PubMed]

11. Danckwardt, S.; Gehring, N.H.; Neu-Yilik, G.; Hundsdoerfer, P.; Pforsich, M.; Frede, U.; Hentze, M.W.; Kulozik, A.E. The prothrombin 3' end formation signal reveals a unique architecture that is sensitive to thrombophilic gain-of-function mutations. *Blood* **2004**, *104*, 428-435. [CrossRef] [PubMed]

12. Nunes, N.M.; Li, W.; Tian, B.; Furger, A. A functional human poly(a) site requires only a potent dse and an a-rich upstream sequence. *EMBO J.* **2010**, *29*, 1523-1536. [CrossRef] [PubMed]

13. Pauws, E.; van Kampen, A.H.; van de Graaf, S.A.; de Vijlder, J.J.; Ris-Stalpers, C. Heterogeneity in poly(A)denylation cleavage sites in mammalian mRNA sequences: Implications for sage analysis. *Nucleic Acids Res.* **2001**, *29*, 1690-1694. [CrossRef] [PubMed]
14. Chen, F.; MacDonald, C.C.; Wilusz, J. Cleavage site determinants in the mammalian poly(A)denylation signal. *Nucleic Acids Res.* **1995**, *23*, 2614–2620. [CrossRef] [PubMed]

15. Li, W.; Laishram, R.S.; Anderson, R.A. The novel poly(A) polymerase Star-PAP is a signal-regulated switch at the 3'-end of mRNAs. *Adv. Biol. Regul.* **2013**, *53*, 64–76. [CrossRef] [PubMed]

16. Dalziel, M.; Nunes, N.M.; Furger, A. Two g-rich regulatory elements located adjacent to and 440 nucleotides downstream of the core poly(a) site of the intronless melanocortin receptor 1 gene are critical for efficient 3' end processing. *Mol. Cell. Biol.* **2007**, *27*, 1568–1580. [CrossRef] [PubMed]

17. Oberg, D.; Fay, J.; Lambkin, H.; Schwartz, S. A downstream poly(A)denylation element in human papillomavirus type 16 L2 encodes multiple GGG motifs and interacts with hnRNP H. *J. Virol.* **2005**, *79*, 9254–9269. [CrossRef] [PubMed]

18. Kandala, D.T.; Mohan, N.; Laishram, R.S. CstF-64 and 3'-UTR cis-element determine Star-PAP specificity for target mRNA selection by excluding PAPα. *Nucleic Acids Res.* **2016**, *44*, 811–823. [CrossRef] [PubMed]

19. Laishram, R.S. Poly(A) polymerase (PAP) diversity in gene expression—Star-PAP vs canonical PAP. *FEBS Lett.* **2014**, *588*, 2185–2197. [CrossRef] [PubMed]

20. Laishram, R.S.; Anderson, R.A. The poly A polymerase Star-PAP controls 3'-end cleavage by promoting CPSF interaction and specificity toward the pre-mRNA. *EMBO J.* **2010**, *29*, 4132–4145. [CrossRef] [PubMed]

21. Mellman, D.L.; Gonzales, M.L.; Song, C.; Barlow, C.A.; Wang, P.; Kendziorski, C.; Anderson, R.A. A PtdIns4,5P2-regulated nuclear poly(A) polymerase controls expression of select mRNAs. *Nature* **2008**, *451*, 1013–1017. [CrossRef] [PubMed]

22. Peart, N.; Wagner, E.J. A distal auxiliary element facilitates cleavage and poly(A)denylation of Dux4 mRNA in the pathogenic haplotype of FSHD. *Hum. Genet.* **2017**, *136*, 1291–1301. [CrossRef] [PubMed]

23. Neve, J.; Patel, R.; Wang, Z.; Louey, A.; Furger, A.M. Cleavage and poly(A)denylation: Ending the message expands gene regulation. *RNA Biol.* **2017**, *14*, 865–890. [CrossRef] [PubMed]

24. Shi, Y.; Manley, J.L. The end of the message: Multiple protein-rna interactions define the mRNA poly(A)denylation site. *Genes Dev.* **2015**, *29*, 889–897. [CrossRef] [PubMed]

25. Chambers, O.; Milenkovic, J.; Praznikar, A.; Tasic, J.F. Computer-based assessment for facioscapulohumeral dystrophy diagnosis. *Comput. Methods Programs Biomed.* **2015**, *120*, 37–48. [CrossRef] [PubMed]

26. Kaufmann, I.; Martin, G.; Friedlein, A.; Langen, H.; Keller, W. Human Fip1 is a subunit of CPSF that binds to U-rich RNA elements and stimulates poly(a) polymerase. *EMBO J.* **2004**, *23*, 616–626. [CrossRef] [PubMed]

27. Shi, Y.; Di Giammartino, D.C.; Taylor, D.; Sarkeshik, A.; Rice, W.J.; Yates, J.R.; Frank, J.; Manley, J.L. Molecular architecture of the human pre-mRNA 3' processing complex. *Mol. Cell* **2009**, *33*, 365–376. [CrossRef] [PubMed]

28. Takagaki, Y.; Manley, J.L. Complex protein interactions within the human poly(A)denylation machinery identify a novel component. *Mol. Cell. Biol.* **2000**, *20*, 1515–1525. [CrossRef] [PubMed]

29. Dominski, Z.; Yang, X.-C.; Purdy, M.; Wagner, E.J.; Marzluff, W.F. A CPSF-73 homologue is required for cell cycle progression but not cell growth and interacts with a protein having features of CPSF-100. *Mol. Cell. Biol.* **2005**, *25*, 1489–1500. [CrossRef] [PubMed]

30. Sullivan, K.D.; Steiniger, M.; Marzluff, W.F. A core complex of CPSF73, CPSF100, and symplekin may form two different cleavage factors for processing of poly(a) and histone mRNAs. *Mol. Cell* **2009**, *34*, 322–332. [CrossRef] [PubMed]

31. Mandel, C.R.; Kaneko, S.; Zhang, H.; Gebauer, D.; Vethantham, V.; Manley, J.L.; Tong, L. Polyadenylation factor CPSF-73 is the pre-mRNA 3'-end-processing endonuclease. *Nature* **2006**, *444*, 953–956. [CrossRef] [PubMed]

32. Ryan, K.; Calvo, O.; Manley, J.L. Evidence that poly(A)denylation factor CPSF-73 is the mRNA 3' processing endonuclease. *RNA* **2004**, *10*, 565–573. [CrossRef] [PubMed]

33. Chan, S.L.; Huppertz, I.; Yao, C.; Weng, L.; Moresco, J.J.; Yates, J.R.; Ule, J.; Manley, J.L.; Shi, Y. CPSF30 and Wdr33 directly bind to AAUAAA in mammalian mRNA 3' processing. *Genes Dev.* **2014**, *28*, 2370–2380. [CrossRef] [PubMed]

34. Clerici, M.; Faini, M.; Aebersold, R.; Jinek, M. Structural insights into the assembly and poly(A) signal recognition mechanism of the human CPSF complex. *eLife* **2017**, *6*, e33111. [CrossRef] [PubMed]

35. Dichtl, B.; Blank, D.; Sadowski, M.; Hubner, W.; Weiser, S.; Keller, W. Yhh1p/Cft1p directly links poly(A) site recognition and RNA polymerase ii transcription termination. *EMBO J.* **2002**, *21*, 4125–4135. [CrossRef] [PubMed]
36. Schönemann, L.; Kuhn, U.; Martin, G.; Schafer, P.; Gruber, A.R.; Keller, W.; Zavolan, M.; Wahle, E. Reconstitution of CPSF active in poly(A)denylation: Recognition of the poly(A)denylation signal by Wdr33. *Genes Dev.* **2014**, *28*, 2381–2393. [CrossRef] [PubMed]

37. Murthy, K.G.; Manley, J.L. The 160-kD subunit of human cleavage-poly(A)denylation specificity factor coordinates pre-mRNA 3'-end formation. *Genes Dev.* **1995**, *9*, 2672–2683. [CrossRef] [PubMed]

38. Gilmartin, G.M.; Nevins, J.R. An ordered pathway of assembly of components required for poly(A)denylation site recognition and processing. *Genes Dev.* **1989**, *3*, 2180–2190. [CrossRef] [PubMed]

39. Takagaki, Y.; Ryner, L.C.; Manley, J.L. Four factors are required for 3'-end cleavage of pre-mRNAs. *Genes Dev.* **1989**, *3*, 1711–1724. [CrossRef] [PubMed]

40. Moreno-Morcillo, M.; Minvielle-Sebastia, L.; Mackereth, C.; Fribourg, S.B. Hexameric architecture of CstF supported by CstF-50 homodimerization domain structure. *RNA* **2011**, *17*, 412–418. [CrossRef] [PubMed]

41. Perez Canadillas, J.M.; Varani, G. Recognition of GU-rich poly(A)denylation regulatory elements by human CstF-64 protein. *EMBO J.* **2003**, *22*, 2821–2830. [CrossRef] [PubMed]

42. Yao, C.; Biesinger, J.; Wan, J.; Weng, L.; Xing, Y.; Xie, X.; Shi, Y. Transcriptome-wide analyses of CstF64-rna interactions in global regulation of mRNA alternative poly(A)denylation. *Proc. Natl. Acad. Sci. USA* **2012**, *109*, 18773–18778. [CrossRef] [PubMed]

43. Kleiman, F.E.; Manley, J.L. The BARD1-CstF-50 interaction links mRNA 3' end formation to DNA damage and tumor suppression. *Cell* **2001**, *104*, 743–753. [CrossRef]

44. Fong, N.; Bentley, D.L. Capping, splicing, and 3' processing are independently stimulated by RNA polymerase II: Different functions for different segments of the CTD. *Genes Dev.* **2001**, *15*, 1783–1795. [CrossRef] [PubMed]

45. Ruepp, M.-D.; Schweingruber, C.; Kleinschmidt, N.; Schümperli, D. Interactions of CstF-64, CstF-77, and symplekin: Implications on localisation and function. *Mol. Biol. Cell* **2011**, *22*, 91–104. [CrossRef] [PubMed]

46. Gruber, A.R.; Martin, G.; Keller, W.; Zavolan, M. Cleavage factor im is a key regulator of 3' UTR length. *RNA Biol.* **2012**, *9*, 1405–1412. [CrossRef] [PubMed]

47. Katahira, J.; Okuzaki, D.; Inoue, H.; Yoneda, Y.; Maehara, K.; Ohkawa, Y. Human TREX component Thoc5 affects alternative poly(A)denylation site choice by recruiting mammalian cleavage factor I. *Nucleic Acids Res.* **2013**, *41*, 7060–7072. [CrossRef] [PubMed]

48. Masamha, C.P.; Xia, Z.; Yang, J.; Albrecht, T.R.; Li, M.; Shyu, A.-B.; Li, W.; Wagner, E.J. CFIm25 links alternative poly(A)denylation to glioblastoma tumor suppression. *Nature* **2014**, *510*, 412–416. [CrossRef] [PubMed]

49. Ruepp, M.-D.; Aringhieri, C.; Vivarelli, S.; Cardinale, S.; Paro, S.; Schümperli, D.; Barabino, S.M.L. Mammalian pre-mRNA 3' end processing factor cf I M 68 functions in mRNA export. *Mol. Biol. Cell* **2009**, *20*, 5211–5223. [CrossRef] [PubMed]

50. Zhu, Y.; Wang, X.; Forouzmand, E.; Jeong, J.; Qiao, F.; Sowd, G.A.; Engelman, A.N.; Xie, X.; Hertel, K.J.; Shi, Y. Molecular mechanisms for CFIm-mediated regulation of mRNA alternative poly(A)denylation. *Mol. Cell* **2018**, *69*, 62–74. [CrossRef] [PubMed]

51. Coseno, M.; Martin, G.; Berger, C.; Gilmartin, G.; Keller, W.; Doublie, S. Crystal structure of the 25 kDa subunit of human cleavage factor I<sub>m</sub>. *Nucleic Acids Res.* **2008**, *36*, 3474–3483. [CrossRef] [PubMed]

52. Ruegsegger, U.; Beyer, K.; Keller, W. Purification and characterization of human cleavage factor im involved in the 3' end processing of messenger RNA precursors. *J. Biol. Chem.* **1996**, *271*, 6107–6113. [CrossRef] [PubMed]

53. Ruegsegger, U.; Blank, D.; Keller, W. Human pre-mRNA cleavage factor I<sub>m</sub> is related to spliceosomal SR proteins and can be reconstituted in vitro from recombinant subunits. *Mol. Cell* **1998**, *1*, 243–253. [CrossRef]

54. Yang, Q.; Coseno, M.; Gilmartin, G.M.; Doublie, S. Crystal structure of a human cleavage factor CFI(m)25/CFI(m)68/RNA complex provides an insight into poly(A) site recognition and RNA looping. *Structure* **2011**, *19*, 368–377. [CrossRef] [PubMed]

55. Brown, K.M.; Gilmartin, G.M. A mechanism for the regulation of pre-mRNA 3' processing by human cleavage factor im. *Mol. Cell* **2003**, *12*, 1467–1476. [CrossRef]

56. Yang, Q.; Gilmartin, G.M.; Doublié, S. Structural basis of ugua recognition by the nudix protein CFI(m)25 and implications for a regulatory role in mRNA 3' processing. *Proc. Natal. Acad. Sci. USA* **2010**, *107*, 10062–10067. [CrossRef] [PubMed]
57. Colgan, D.F.; Manley, J.L. Mechanism and regulation of mRNA poly(A)denylation. *Genes Dev.* **1997**, *11*, 2755-2766. [CrossRef] [PubMed]

58. Mandel, C.R.; Bai, Y.; Tong, L. Protein factors in pre-mRNA 3'-end processing. *Cell. Mol. Life Sci. CMLS* **2008**, *65*, 1099-1122. [CrossRef] [PubMed]

59. Zhao, J.; Hyman, L.; Moore, C. Formation of mRNA 3' ends in eukaryotes: Mechanism, regulation, and interrelationships with other steps in mRNA synthesis. *Microbiol. Mol. Biol. Rev. MMBR* **1999**, *63*, 405-445. [PubMed]

60. Raabe, T.; Bollum, F.J.; Manley, J.L. Primary structure and expression of bovine poly(A) polymerase. *Nature* **1991**, *353*, 229-234. [CrossRef] [PubMed]

61. Wahle, E. Purification and characterization of a mammalian poly(A)denylate polymerase involved in the 3' end processing of messenger RNA precursors. *J. Biol. Chem.* **1991**, *266*, 3131-3139. [PubMed]

62. Wahle, E. A novel poly(A)-binding protein acts as a specificity factor in the second phase of messenger RNA poly(A)denylation. *Cell* **1991**, *66*, 759-768. [CrossRef]

63. Bienroth, S.; Keller, W.; Wahle, E. Assembly of a processive messenger RNA poly(A)denylation complex. *EMBO J.* **1993**, *12*, 585-594. [PubMed]

64. Kerwitz, Y.; Kühn, U.; Lilie, H.; Knoth, A.; Scheuermann, T.; Friedrich, H.; Schwarz, E.; Wahle, E. Stimulation of poly(A) polymerase through a direct interaction with the nuclear poly(A) binding protein allosterically regulated by rna. *EMBO J.* **2003**, *22*, 3705-3714. [CrossRef] [PubMed]

65. Kuhn, U.; Wahle, E. Structure and function of poly(A) binding proteins. *Biochim. Biophys. Acta* **2004**, *1678*, 67-84. [CrossRef] [PubMed]

66. Apponi, L.H.; Leung, S.W.; Williams, K.R.; Valentini, S.R.; Corbett, A.H.; Pavlath, G.K. Loss of nuclear poly(A)-binding protein 1 causes defects in myogenesis and mRNA biogenesis. *Hum. Mol. Genet.* **2010**, *19*, 1058-1065. [CrossRef] [PubMed]

67. Chen, Z.; Li, Y.; Krug, R.M. Influenza a virus ns1 protein targets poly(A)-binding protein II of the cellular 3'-end processing machinery. *EMBO J.* **1999**, *18*, 2273-2283. [CrossRef] [PubMed]

68. Lee, Y.J.; Glaunsinger, B.A. Aberrant herpesvirus-induced poly(A)denylation correlates with cellular messenger RNA destruction. *PLoS Biol.* **2009**, *7*, e1000107. [CrossRef] [PubMed]

69. Bentley, D.L. Rules of engagement: Co-transcriptional recruitment of pre-mRNA processing factors. *Curr. Opin. Cell Biol.* **2005**, *17*, 251-256. [CrossRef] [PubMed]

70. Hirose, Y.; Manley, J.L. RNA polymerase II and the integration of nuclear events. *Genes Dev.* **2000**, *14*, 1415-1429. [PubMed]

71. Hirose, Y.; Manley, J.L. Rna polymerase II is an essential mRNA poly(A)denylation factor. *Nature* **1998**, *395*, 93-96. [CrossRef] [PubMed]

72. McCracken, S.; Fong, N.; Yankulov, K.; Ballantyne, S.; Pan, G.; Greenblatt, J.; Patterson, S.D.; Wickens, M.; Bentley, D.L. The C-terminal domain of RNA polymerase II couples mRNA processing to transcription. *Nature* **1997**, *385*, 357-361. [CrossRef] [PubMed]

73. Sadowski, M.; Dichtl, B.; Hübner, W.; Keller, W. Independent functions of yeast Pcf11p in pre-mRNA 3' end processing and in transcription termination. *EMBO J.* **2003**, *22*, 2167-2177. [CrossRef] [PubMed]

74. McCracken, S.; Fong, N.; Rosonina, E.; Yankulov, K.; Brothers, G.; Siderovski, D.; Hessel, A.; Foster, S.; Shuman, S.; Bentley, D.L. 5'-capping enzymes are targeted to pre-mRNA by binding to the phosphorylated carboxy-terminal domain of RNA polymerase II. *Genes Dev.* **1997**, *11*, 3306-3318. [CrossRef] [PubMed]

75. Flaherty, S.M.; Fortes, P.; Izaurralde, E.; Mattaj, I.W.; Gilmartin, G.M. Participation of the nuclear cap binding complex in pre-mRNA 3' processing. *Proc. Natl. Acad. Sci. USA* **1997**, *94*, 11893-11898. [CrossRef] [PubMed]

76. Jiao, X.; Chang, J.H.; Kilic, T.; Tong, L.; Kiledjian, M. A mammalian pre-mRNA 5' end capping quality control mechanism and an unexpected link of capping to pre-mRNA processing. *Mol. Cell* **2013**, *50*, 104-115. [CrossRef] [PubMed]

77. Danckwardt, S.; Hentze, M.W.; Kulozik, A.E. 3' end mRNA processing: Molecular mechanisms and implications for health and disease. *EMBO J.* **2008**, *27*, 482-498. [CrossRef] [PubMed]

78. Keller, W.; Bienroth, S.; Lang, K.M.; Christofori, G. Cleavage and poly(A)denylation factor cpf specifically interacts with the pre-mRNA 3' processing signal AAUAAA. *EMBO J.* **1991**, *10*, 4241-4249. [PubMed]

79. Dantonel, J.C.; Murthy, K.G.; Manley, J.L.; Tora, L. Transcription factor tfid recruits factor CPSF for formation of 3' end of mRNA. *Nature* **1997**, *389*, 399-402. [CrossRef] [PubMed]
80. Takagaki, Y.; Manley, J.L. RNA recognition by the human poly(A)denylation factor CstF. *Mol. Cell. Biol.* **1997**, *17*, 3907–3914. [CrossRef] [PubMed]

81. Kim, M.; Krogan, N.J.; Vasiljeva, L.; Rando, O.J.; Nedea, E.; Greenblatt, J.F.; Buratowski, S. The yeast rat1 exonuclease promotes transcription termination by RNA polymerase II. *Nature* **2004**, *432*, 517–522. [CrossRef] [PubMed]

82. West, S.; Gromak, N.; Proudfoot, N.J. Human 5' → 3' exonuclease Xrn2 promotes transcription termination at co-transcriptional cleavage sites. *Nature* **2004**, *432*, 522–525. [CrossRef] [PubMed]

83. Mangus, D.A.; Evans, M.C.; Jacobson, A. Poly(A)-binding proteins: Multifunctional scaffolds for the post-transcriptional control of gene expression. *Genome Biol.* **2003**, *4*, 223. [CrossRef] [PubMed]

84. Millevoi, S.; Vagner, S. Molecular mechanisms of eukaryotic pre-mRNA 3' end processing regulation. *Nucleic Acids Res.* **2010**, *38*, 2757–2774. [CrossRef] [PubMed]

85. Derti, A.; Garrett-Engele, P.; Macisaac, K.D.; Stevens, R.C.; Sriram, S.; Chen, R.; Rohl, C.A.; Johnson, J.M.; Babak, T. A quantitative atlas of poly(A)denylation in five mammals. *Genome Res.* **2012**, *22*, 1173–1183. [CrossRef] [PubMed]

86. Hoque, M.; Ji, Z.; Zheng, D.; Luo, W.; Li, W.; You, B.; Park, J.Y.; Yehia, G.; Tian, B. Analysis of alternative cleavage and poly(A)denylation by 3' region extraction and deep sequencing. *Nat. Methods* **2013**, *10*, 133–139. [CrossRef] [PubMed]

87. Sandberg, R.; Neilson, J.R.; Sarma, A.; Sharp, P.A.; Burge, C.B. Proliferating cells express mRNAs with shortened 3' untranslated regions and fewer microrna target sites. *Science* **2008**, *320*, 1643–1647. [CrossRef] [PubMed]

88. Ji, Z.; Lee, J.Y.; Pan, Z.; Jiang, B.; Tian, B. Progressive lengthening of 3' untranslated regions of mRNAs by alternative poly(A)denylation during mouse embryonic development. *Proc. Natl. Acad. Sci. USA* **2009**, *106*, 7028–7033. [CrossRef] [PubMed]

89. Denome, R.M.; Cole, C.N. Patterns of poly(A)denylation site selection in gene constructs containing multiple poly(A)denylation signals. *Mol. Cell. Biol.* **1988**, *8*, 4829–4839. [CrossRef] [PubMed]

90. Pinto, P.A.; Henriques, T.; Freitas, M.O.; Martins, T.; Domingues, R.G.; Wyrzykowska, P.S.; Coelho, P.A.; Carmo, A.M.; Sunkel, C.E.; Proudfoot, N.J.; et al. Rna polymerase II kinetics in polo poly(A)denylation signal selection. *EMBO J.* **2011**, *30*, 2431–2444. [CrossRef] [PubMed]

91. Peterson, M.L.; Bertolino, S.; Davis, F. An RNA polymerase pause site is associated with the immunoglobulin mus poly(A) site. *Mol. Cell. Biol.* **2002**, *22*, 5606–5615. [CrossRef] [PubMed]

92. Kellner, W.A.; Bell, J.S.; Vertino, P.M. GC skew defines distinct RNA polymerase pause sites in CpG island promoters. *Genome Res.* **2015**, *25*, 1600–1609. [CrossRef] [PubMed]

93. Wood, A.J.; Schulz, R.; Woodfine, K.; Koltowska, K.; Beechey, C.V.; Peters, J.; Bourc'his, D.; Oakey, R.J. Regulation of alternative poly(A)denylation by genomic imprinting. *Genes Dev.* **2008**, *22*, 1141–1146. [CrossRef] [PubMed]

94. Takagaki, Y.; Seipelt, R.L.; Peterson, M.L.; Manley, J.L. The poly(A)denylation factor CstF-64 regulates alternative processing of igm heavy chain pre-mRNA during b cell differentiation. *Cell* **1996**, *87*, 941–952. [CrossRef]

95. Hardy, J.G.; Norbury, C.J. Cleavage factor im (CFIm) as a regulator of alternative poly(A)denylation. *Biochem. Soc. Trans.* **2016**, *44*, 1051–1057. [CrossRef] [PubMed]

96. Martin, G.; Gruber, A.R.; Keller, W.; Zavolan, M. Genome-wide analysis of pre-mRNA 3' end processing reveals a decisive role of human cleavage factor I in the regulation of 3' UTR length. *Cell Rep.* **2012**, *1*, 753–763. [CrossRef] [PubMed]

97. Li, W.; You, B.; Hoque, M.; Zheng, D.; Luo, W.; Ji, Z.; Park, J.Y.; Gunderson, S.I.; Kalsotra, A.; Manley, J.L.; et al. Systematic profiling of poly(A)+ transcripts modulated by core 3' end processing and splicing factors reveals regulatory rules of alternative cleavage and poly(A)denylation. *PLoS Genet.* **2015**, *11*, e1005166. [CrossRef] [PubMed]

98. Lee, C.Y.; Chen, L. Alternative poly(A)denylation sites reveal distinct chromatin accessibility and histone modification in human cell lines. *Bioinformatics* **2013**, *29*, 1713–1717. [CrossRef] [PubMed]

99. Huang, H.; Chen, J.; Liu, H.; Sun, X. The nucleosome regulates the usage of poly(A)denylation sites in the human genome. *BMC Genom.* **2013**, *14*, 912. [CrossRef] [PubMed]

100. Kaida, D.; Berg, M.G.; Younis, I.; Kasim, M.; Singh, L.N.; Wan, L.; Dreyfuss, G. U1 snrnp protects pre-mRNAs from premature cleavage and poly(A)denylation. *Nature* **2010**, *468*, 664–668. [CrossRef] [PubMed]
101. Berg, M.G.; Singh, L.N.; Younis, I.; Liu, Q.; Pinto, A.M.; Kaida, D.; Zhang, Z.; Cho, S.; Sherrill-Mix, S.; Wan, L.; et al. U1 snrnp determines mRNA length and regulates isoform expression. *Cell* **2012**, *150*, 53–64. [CrossRef] [PubMed]

102. Gruber, A.R.; Martin, G.; Muller, P.; Schmidt, A.; Gruber, A.J.; Gumienny, R.; Mittal, N.; Jayachandran, R.; Pieters, J.; Keller, W.; et al. Global 3' UTR shortening has a limited effect on protein abundance in proliferating T cells. *Nat. Commun.* **2014**, *5*, 5465. [CrossRef] [PubMed]

103. Spies, N.; Burge, C.B.; Bartel, D.P. 3' UTR isoform choice has limited influence on the stability and translational efficiency of most mRNAs in mouse fibroblasts. *Genome Res.* **2013**, *23*, 2078–2090. [CrossRef] [PubMed]

104. Nam, J.W.; Rissland, O.S.; Koppstein, D.; Abreu-Goodger, C.; Jan, C.H.; Agarwal, V.; Yildirim, M.A.; Rodriguez, A.; Bartel, D.P. Global analyses of the effect of different cellular contexts on microrna targeting. *Mol. Cell* **2014**, *53*, 1031–1043. [CrossRef] [PubMed]

105. Neve, J.; Burger, K.; Li, W.; Hoque, M.; Patel, R.; Tian, B.; Gullerova, M.; Furger, A. Subcellular RNA profiling links splicing and nuclear dicer1 to alternative cleavage and poly(A)denylation. *Genome Res.* **2016**, *26*, 24–35. [CrossRef] [PubMed]

106. Mayr, C. Evolution and biological roles of alternative 3' UTRs. *Trends Cell Biol.* **2016**, *26*, 227–237. [CrossRef] [PubMed]

107. Chiabudini, M.; Conz, C.; Reckmann, F.; Rospert, S. Ribosome-associated complex and SSB are required for translational repression induced by polylysine segments within nascent chains. *Mol. Cell. Biol.* **2012**, *32*, 4769–4779. [CrossRef] [PubMed]

108. Klauer, A.A.; van Hoof, A. Degradation of mRNAs that lack a stop codon: A decade of nonstop progress. *Wiley Interdiscip. Rev. *2012*, *3*, 649–660. [CrossRef] [PubMed]

109. Mbita, Z.; Meyer, M.; Skepu, A.; Hosie, M.; Rees, J.; Dlamini, Z. De-regulation of the RBBP6 isoform 3/DWNN in human cancers. *Mol. Cell. Biochem.* **2012**, *362*, 249–262. [CrossRef] [PubMed]

110. Di Giammartino, D.C.; Li, W.; Ogami, K.; Yashinskie, J.J.; Hoque, M.; Tian, B.; Manley, J.L. RBBP6 isoforms regulate the human poly(A)denylation machinery and modulate expression of mRNAs with AU-rich 3' UTRs. *Genes Dev.* **2014**, *28*, 2248–2260. [CrossRef] [PubMed]

111. Ji, Z.; Tian, B. Reprogramming of 3' untranslated regions of mRNAs by alternative poly(A)denylation in generation of pluripotent stem cells from different cell types. *PLoS ONE* **2009**, *4*, e8419. [CrossRef] [PubMed]

112. Alt, F.W.; Bothwell, A.L.; Knapp, M.; Siden, E.; Mather, E.; Koshland, M.; Baltimore, D. Synthesis of secreted and membrane-bound immunoglobulin mu heavy chains is directed by mRNAs that differ at their 3' ends. *Cell* **1980**, *20*, 293–301. [CrossRef]

113. Peterson, M.L. Mechanisms controlling production of membrane and secreted immunoglobulin during B cell development. *Immunol. Res.* **2007**, *37*, 33–46. [CrossRef] [PubMed]

114. Raz, V.; Riaz, M.; Tatum, Z.; Kielbasa, S.M.; Hoen, P.A.C. The distinct transcriptomes of slow and fast adult muscles are delineated by noncoding RNAs. *FASEB J.* **2018**. [CrossRef] [PubMed]

115. Boutet, S.C.; Cheung, T.H.; Quach, N.L.; Liu, L.; Prescott, S.L.; Edalati, A.; Iori, K.; Rando, T.A. Alternative poly(A)denylation mediates microrna regulation of muscle stem cell function. *Cell Stem Cell* **2012**, *10*, 327–336. [CrossRef] [PubMed]

116. Vorobyov, E.; Horst, J. Expression of two protein isoforms of PAX7 is controlled by competing cleavage-poly(A)denylation and splicing. *Gene* **2004**, *342*, 107–112. [CrossRef] [PubMed]

117. Vest, K.E.; Paskavitz, A.L.; Lee, J.B.; Padilla-Benavides, T. Dynamic changes in copper homeostasis and post-transcriptional regulation of Atp7a during myogenic differentiation. *Metallomics* **2018**, *10*, 309–322. [CrossRef] [PubMed]

118. Kim, D.J.; Oh, B.; Kim, Y.Y. Splicing factor ASF/SF2 and transcription factor PPAR-gamma cooperate to directly regulate transcription of uncoupling protein-3. *Biochem. Biophys. Res. Commun.* **2009**, *378*, 877–882. [CrossRef] [PubMed]

119. Joubert, R.; Metayer Coustard, S.; Swennen, Q.; Sibut, V.; Crochet, S.; Cailleau-Audouin, E.; Buyse, J.; Decuypere, E.; Wrutniak-Cabello, C.; Cabello, G.; et al. The beta-adrenergic system is involved in the regulation of the expression of avian uncoupling protein in the chicken. *Domest. Anim. Endocrinol.* **2010**, *38*, 115–125. [CrossRef] [PubMed]

120. Yoon, M.S. Mtor as a key regulator in maintaining skeletal muscle mass. *Front. Physiol.* **2017**, *8*, 788. [CrossRef] [PubMed]
121. Chang, J.W.; Zhang, W.; Yeh, H.S.; de Jong, E.P.; Jun, S.; Kim, K.H.; Bae, S.S.; Beckman, K.; Hwang, T.H.; Kim, K.S.; et al. mRNA 3'-UTR shortening is a molecular signature of mTORC1 activation. *Nat. Commun.* **2015**, *6*, 7218. [CrossRef] [PubMed]

122. Zhang, Y.; King, O.D.; Rahimov, F.; Jones, T.I.; Ward, C.W.; Kerr, J.P.; Liu, N.; Emerson, C.P., Jr.; Kunkel, L.M.; Partridge, T.A.; et al. Human skeletal muscle xenograft as a new preclinical model for muscle disorders. *Hum. Mol. Genet.* **2014**, *23*, 3180–3188. [CrossRef] [PubMed]

123. Monks, D.A.; Holmes, M.M. Androgen receptors and muscle: A key mechanism underlying life history trade-offs. *J. Comp. Physiol.* **2018**, *204*, 51–60. [CrossRef] [PubMed]

124. Hu, R.; Dunn, T.A.; Wei, S.; Isharwal, S.; Veltri, R.W.; Humphreys, E.; Han, M.; Partin, A.W.; Vessella, R.L.; Isaacs, W.B.; et al. Ligand-independent androgen receptor variants derived from splicing of cryptic exons signify hormone-refractory prostate cancer. *Cancer Res.* **2009**, *69*, 16–22. [CrossRef] [PubMed]

125. Tavanez, J.P.; Calado, P.; Braga, J.; Lafarga, M.; Carmo-Fonseca, M. In vivo aggregation properties of the nuclear poly(A)-binding protein pabpn1. *RNA* **2005**, *11*, 752–762. [CrossRef] [PubMed]

126. Jenal, M.; Elkon, R.; Loayza-Puch, F.; van Haaften, G.; Kuhn, U.; Menzies, F.M.; Oude Vrielink, J.A.; Bos, A.J.; Drost, J.; Rooijers, K.; et al. The poly(A)-binding protein nuclear 1 suppresses alternative cleavage and poly(A)denylation sites. *Cell* **2012**, *149*, 538–553. [CrossRef] [PubMed]

127. De Klerk, E.; Venema, A.; Anvar, S.Y.; Goeman, J.J.; Hu, O.; Trollet, C.; Dickson, G.; den Dunnen, J.T.; van der Maarel, S.M.; Raz, V.; et al. Poly(A) binding protein nuclear 1 levels affect alternative poly(A)denylation. *Nucleic Acids Res.* **2012**, *40*, 9089–9101. [CrossRef] [PubMed]

128. Banerjee, A.; Apponi, L.H.; Pavlath, G.K.; Corbett, A.H. Pabpn1: Molecular function and muscle disease. *FEBS J.* **2013**, *280*, 4230–4250. [CrossRef] [PubMed]

129. Batra, R.; Charizanis, K.; Manchanda, M.; Mohan, A.; Li, M.; Finn, D.J.; Goodwin, M.; Zhang, C.; Sobczak, K.; Thornton, C.A.; et al. Loss of mbnl leads to disruption of developmentally regulated alternative poly(A)denylation in rna-mediated disease. *Mol. Cell* **2014**, *56*, 311–322. [CrossRef] [PubMed]

130. Udd, B.; Krahe, R. The myotonic dystrophies: Molecular, clinical, and therapeutic challenges. *Lancet* **2012**, *11*, 891–905. [CrossRef]

131. Brook, J.D.; McCurrach, M.E.; Harley, H.G.; Buckler, A.J.; Church, D.; Aburatani, H.; Hunter, K.; Stanton, V.P.; Thirion, J.P.; Hudson, T.; et al. Molecular basis of myotonic dystrophy: Expansion of a trinucleotide (CTG) repeat at the 3' end of a transcript encoding a protein kinase family member. *Cell* **1992**, *68*, 799–808. [CrossRef]

132. Tian, B.; White, R.J.; Xia, T.; Welle, S.; Turner, D.H.; Mathews, M.B.; Thornton, C.A. Expanded CUG repeat RNAs form hairpins that activate the double-stranded rna-dependent protein kinase PKR. *RNA* **2000**, *6*, 79–87. [CrossRef] [PubMed]

133. Pettersson, O.J.; Aagaard, L.; Jensen, T.G.; Damgaard, C.K. Molecular mechanisms in DM1—A focus on foci. *Nucleic Acids Res.* **2015**, *43*, 2433–2441. [CrossRef] [PubMed]

134. Ferreboeuf, M.; Mariot, V.; Furling, D.; Butler-Browne, G.; Mouly, V.; Dumonceaux, J. Nuclear protein spreading: Implication for pathophysiology of neuromuscular diseases. *Hum. Mol. Genet.* **2014**, *23*, 4125–4133. [CrossRef] [PubMed]

135. Gao, Y.; Guo, X.; Santostefano, K.; Wang, Y.; Reid, T.; Zeng, D.; Terada, N.; Ashizawa, T.; Xia, G. Genome therapy of myotonic dystrophy type 1 iPS cells for development of autologous stem cell therapy. *Mol. Ther.* **2016**, *24*, 1378–1387. [CrossRef] [PubMed]

136. Chen, J.C.; King, O.D.; Zhang, Y.; Clayton, N.P.; Spencer, C.; Wentworth, B.M.; Emerson, C.P., Jr.; Wagner, K.R. Morpholino-mediated knockdown of DUX4 toward facioscapulohumeral muscular dystrophy therapeutics. *Mol. Ther.* **2016**, *24*, 1405–1411. [CrossRef] [PubMed]

137. Marsollier, A.C.; Ciszewski, L.; Mariot, V.; Popplewell, L.; Voit, T.; Dickson, G.; Dumonceaux, J. Antisense targeting of 3' end elements involved in DUX4 mRNA processing is an efficient therapeutic strategy for facioscapulohumeral dystrophy: A new gene-silencing approach. *Hum. Mol. Genet.* **2016**, *25*, 1468–1478. [CrossRef] [PubMed]

138. Tawil, R. Facioscapulohumeral muscular dystrophy. *Neurotherapeutics* **2008**, *5*, 601–606. [CrossRef] [PubMed]

139. Tasca, G.; Monforte, M.; Iannaccone, E.; Laschena, F.; Ottaviani, P.; Leoncini, E.; Boccia, S.; Galluzzi, G.; Pelliccioni, M.; Masciullo, M.; et al. Upper girdle imaging in facioscapulohumeral muscular dystrophy. *PLoS ONE* **2014**, *9*, e100292. [CrossRef] [PubMed]
140. Wijmenga, C.; Hewitt, J.E.; Sandkuijl, L.A.; Clark, L.N.; Wright, T.J.; Dauwerse, H.G.; Gruter, A.M.; Hofker, M.H.; Moerer, P.; Williamson, R.; et al. Chromosome 4q DNA rearrangements associated with facioscapulohumeral muscular dystrophy. *Nat. Genet.* **1992**, *2*, 26–30. [CrossRef] [PubMed]

141. Lemmers, R.J.; van der Vliet, P.J.; Klooster, R.; Sacconi, S.; Camano, P.; Dauwerse, J.G.; Snider, L.; Straasheijm, K.R.; van Ommen, G.J.; Padberg, G.W.; et al. A unifying genetic model for facioscapulohumeral muscular dystrophy. *Science* **2010**, *329*, 1650–1653. [CrossRef] [PubMed]

142. Dixit, M.; Ansseau, E.n.; Tassin, A.; Winokur, S.; Shi, R.; Qian, H.; Sauvage, S.b.; Mattéotti, C.; van Acker, A.M.; Leo, O.; et al. DUX4, a candidate gene of facioscapulohumeral muscular dystrophy, encodes a transcriptional activator of PITX1. *Proc. Natl. Acad. Sci. USA* **2007**, *104*, 18157–18162. [CrossRef] [PubMed]

143. Lemmers, R.J.; Wohlgemuth, M.; Frants, R.R.; Padberg, G.W.; Morava, E.; van der Maarel, S.M. Contractions of D4Z4 on 4qB subtelomeres do not cause facioscapulohumeral muscular dystrophy. *Am. J. Hum. Genet.* **2004**, *75*, 1124–1130. [CrossRef] [PubMed]

144. Lemmers, R.J.; Tawil, R.; Petek, L.M.; Balog, J.; Block, G.J.; Santen, G.W.; Amell, A.M.; van der Vliet, P.J.; Almomani, R.; Straasheijm, K.R.; et al. Digenic inheritance of an smchd1 mutation and an FSHD-permissive D4Z4 allele causes facioscapulohumeral muscular dystrophy type 2. *Nat. Genet.* **2012**, *44*, 1370–1374. [CrossRef] [PubMed]

145. Van den Boogaard, M.L.; Lemmers, R.; Balog, J.; Wohlgemuth, M.; Auranen, M.; Mitsuhashi, S.; van der Vliet, P.J.; Straasheijm, K.R.; van den Akker, R.F.P.; Kriek, M.; et al. Mutations in DNMT3b modify epigenetic repression of the D4Z4 repeat and the penetrance of facioscapulohumeral dystrophy. *Am. J. Hum. Genet.* **2016**, *98*, 1020–1029. [CrossRef] [PubMed]

146. Caruso, N.; Herberth, B.; Bartoli, M.; Puppo, F.; Dumonceaux, J.; Zimmermann, A.; Denadai, S.; Lebosse, M.; Roche, S.; Geng, L.; et al. Deregulation of the protocadherin gene FAT1 alters muscle shapes: Implications for the pathogenesis of facioscapulohumeral dystrophy. *PLoS Genet.* **2013**, *9*, e1003550. [CrossRef] [PubMed]

147. Mariot, V.; Roche, S.; Hourde, C.; Portilho, D.; Sacconi, S.; Puppo, F.; Duguez, S.; Rameau, P.; Caruso, N.; Delezoide, A.L.; et al. Correlation between low FAT1 expression and early affected muscle in facioscapulohumeral muscular dystrophy. *Ann. Neurol.* **2015**, *78*, 387–400. [CrossRef] [PubMed]

148. Ferreboeuf, M.; Mariot, V.; Bessieres, B.; Vasiljevic, A.; Attie-Bitach, T.; Collardeau, S.; Moreire, J.; Roche, S.; Magdinier, F.; Robin-Ducellier, J.; et al. DUX4 and DUX4 downstream target genes are expressed in fetal FSHD muscles. *Hum. Mol. Genet.* **2014**, *23*, 171–181. [CrossRef] [PubMed]

149. Broucqsault, N.; Moreire, J.; Gaillard, M.C.; Dumonceaux, J.; Torrents, J.; Salort-Campana, E.; Maues de Paula, A.; Bartoli, M.; Fernandez, C.; Chesnais, A.L.; et al. Dysregulation of 4q35- and muscle-specific genes in fetuses with a short D4Z4 array linked to facio-scapulo-humeral dystrophy. *Hum. Mol. Genet.* **2013**, *22*, 4206–4214. [CrossRef] [PubMed]

150. Snider, L.; Asawachaicharn, A.; Tyler, A.E.; Geng, L.N.; Petek, L.M.; Maves, L.; Miller, D.G.; Lemmers, R.J.L.F.; Winokur, S.T.; Tawil, R.; et al. Rna transcripts, miRNA-sized fragments and proteins produced from D4Z4 units: New candidates for the pathophysiology of facioscapulohumeral dystrophy. *Hum. Mol. Genet.* **2009**, *18*, 2414–2430. [CrossRef] [PubMed]

151. Gaillard, M.C.; Roche, S.; Dion, C.; Tasmadjian, A.; Bouget, G.; Salort-Campana, E.; Vovan, C.; Chaix, C.; Broucqsault, N.; Moreire, J.; et al. Differential DNA methylation of the D4Z4 repeat in patients with FSHD and asymptomatic carriers. *Neurology* **2014**, *83*, 733–742. [CrossRef] [PubMed]

152. Yao, Z.; Snider, L.; Balog, J.; Lemmers, R.J.; Van Der Maarel, S.M.; Tawil, R.; Tapscott, S.J. DUX4-induced gene expression is the major molecular signature in FSHD skeletal muscle. *Hum. Mol. Genet.* **2014**, *23*, 5342–5352. [CrossRef] [PubMed]

153. Xia, G.; Gao, Y.; Jin, S.; Subramony, S.H.; Terada, N.; Ranum, L.P.; Swanson, M.S.; Ashizawa, T. Genome modification leads to phenotype reversal in human myotonic dystrophy type 1 induced pluripotent stem cell-derived neural stem cells. *Stem Cells* **2015**, *33*, 1829–1838. [CrossRef] [PubMed]

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