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YSCDB 1551 110 ARTICLE IN PRESS

Seminars in Cell & Developmental Biology xxx (2014) xxxxxx

Contents lists available at ScienceDirect

Seminars in Cell & Developmental Biology

journal homepage: www.elsevier.com/locate/semcdb

1 Review

2 Quality control of mRNP biogenesis: Networking at the

3 transcription site
4 Q1 Andrea B. Eberle, Neus Visa
5 Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, SE-106 91 Stockholm, Sweden
6

7
19 a r t i c l e i n f o a b s t r a c t
8
9 Article history: Eukaryotic cells carry out quality control (QC) over the processes of RNA biogenesis to inactivate or elim-
10 Available online xxx inate defective transcripts, and to avoid their production. In the case of protein-coding transcripts, the
11 quality controls can sense defects in the assembly of mRNAprotein complexes, in the processing of the
12 Keywords: precursor mRNAs, and in the sequence of open reading frames. Different types of defect are monitored by
13 Exosome different specialized mechanisms. Some of them involve dedicated factors whose function is to identify
14 XRN2
faulty molecules and target them for degradation. Others are the result of a more subtle balance in the
15 UPF proteins
kinetics of opposing activities in the mRNA biogenesis pathway. One way or another, all such mechanisms
16 Nuclear retention
17 Transcription
hinder the expression of the defective mRNAs through processes as diverse as rapid degradation, nuclear
18 Degradation retention and transcriptional silencing. Three major degradation systems are responsible for the destruc-
tion of the defective transcripts: the exosome, the 5 3 exoribonucleases, and the nonsense-mediated
mRNA decay (NMD) machinery. This review summarizes recent ndings on the cotranscriptional qual-
ity control of mRNA biogenesis, and speculates that a proteinprotein interaction network integrates
multiple mRNA degradation systems with the transcription machinery.
2014 Published by Elsevier Ltd.

20 Contents

21 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
22 1.1. Nuclear mechanisms of quality control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
23 2. The exosome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
24 2.1. Nuclear surveillance of mRNA biogenesis by the exosome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
25 2.2. The recruitment of the exosome to nascent transcripts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
26 3. QC of pre-mRNP assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
27 4. QC of 5 -end capping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
28 5. QC of splicing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
29 5.1. Retention at the transcription site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
30 5.2. Degradation of unspliced transcripts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
31 5.3. Splicing and transcription downregulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
32 5.4. Splicing and nonsense mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
33 6. QC of 3 end formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
34 7. Integration of mRNA decay systems at the site of transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
35 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
36 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Corresponding author. Tel.: +46 8 164111; fax: +46 8 166488.

E-mail address: neus.visa@su.se (N. Visa).

http://dx.doi.org/10.1016/j.semcdb.2014.03.033
1084-9521/ 2014 Published by Elsevier Ltd.

Please cite this article in press as: Eberle AB, Visa N. Quality control of mRNP biogenesis: Networking at the transcription site. Semin
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37 1. Introduction 2. The exosome 98

38 Nascent pre-mRNAs synthesized by RNA polymerase II (Pol-II) The exosome is a multiprotein complex with ribonucleolytic 99

39 become rapidly capped at the 5 end and associated with a plethora activity and is a major player in the QC of mRNA biogenesis in 100

40 of RNA-binding proteins that package the pre-mRNA into func- the cell nucleus (reviewed by [17,18]). The structure of the exo- 101

41 tionally competent pre-mRNP complexes [1]. The pre-mRNAs are some is highly conserved throughout evolution, and exosomes from 102

42 processed in the nucleus and the mature mRNPs are exported to archaea and metazoans have a similar organization, based on a 103

43 the cytoplasm. Mutations in the DNA or errors in the packaging nine-subunit core (reviewed in [19,20]). The core subunits of the 104

44 or processing of the transcripts can lead to the formation of faulty eukaryotic exosome are necessary for the proper function of the 105

45 mRNPs whose translation could result in the synthesis of poten- exosome, but are catalytically inactive. The activity of the complex 106

46 tially harmful proteins or interfere with regular RNA processing or is provided by two hydrolytic RNases, RRP6 and RRP44/DIS3, that 107

47 RNP assembly. Eukaryotes have evolved sophisticated QC systems associate with the core in organisms as diverse as yeast, insects 108

48 that deal with those transcripts through a variety of mechanisms, to and humans [2124]. A third active subunit, DIS3L, has recently 109

49 minimize the risks associated with the expression of faulty mRNPs. been identied in human cells [25,26]. RRP6 and DIS3L are 3 5 110

50 Collectively, the QC systems can sense defects in the assembly of exoRNases, whereas RRP44/DIS3 has both endoRNase and 3 5 111

51 the (pre)mRNP complexes, in the processing of the transcripts, and exoRNase activities [27,28]. 112

52 in the sequence of the open reading frames. Moreover, QC mech- The exosome is the major 3 5 RNase in the cell nucleus, where 113

53 anisms also eliminate pervasive transcripts that are synthesized it is responsible for the processing of many non-coding RNAs (ncR- 114

54 throughout a large fraction of the genome [2]. The synthesis and NAs), including rRNAs, tRNAs, snRNAs and snoRNAs, and for the 115

55 turnover of such pervasive transcripts have recently been reviewed degradation of different types of cryptic and pervasive transcripts 116

56 by Jensen et al. [3]. Here, we focus on the QC of protein-coding (reviewed by [2,3,29]). The molecular mechanisms by which dif- 117

57 transcripts in the cell nucleus. ferent RNA species are specically recognized by the exosome, and 118

either processed in a controlled manner or totally degraded, are still 119

unclear. What is clear is that the functions of the exosome depend 120

58 1.1. Nuclear mechanisms of quality control strongly on a multitude of exosome-interacting factors, adaptors or 121

cofactors, that target the exosome to specic cellular locations, reg- 122

59 The nuclear mechanisms of QC monitor every step in gene ulate its activity, and contribute to the recognition of the different 123

60 expression, starting with the production of the transcript by Pol- RNA substrates (reviewed by [17,30]). 124

61 II and nishing with the export of the mRNP through the nuclear An additional level of complexity in the functional study of the 125

62 pore complex (NPC) (Fig. 1). Most of the pre-mRNA processing steps exosome arises from the fact that individual exosome subunits may 126

63 occur when the transcripts are still attached to the gene locus by act independently of the entire complex. Results obtained in studies 127

64 the transcriptional machinery, and QC mechanisms act at the site of individual subunits are often extrapolated to the entire exosome, 128

65 of transcription to take care of faulty transcripts that otherwise based on the nding that the exosome is a multi-subunit complex 129

66 could be overseen by later control mechanisms. One benet of an with conserved composition and stoichiometry [31,32]. However, 130

67 early control mechanism at the transcription site is that no energy there is evidence that the exosome RNases exist in a multitude of 131

68 is wasted in the processing and transport of non-functional tran- subcomplexes and act independently of the core [33]. For example, 132

69 scripts. a mutant Rrp6p that cannot interact with the exosome core in S. 133

70 In general, three strategies are used in the cell nucleus to deal cerevisiae is still able to support pre-rRNA processing reactions [34], 134

71 with faulty transcripts: retention of transcripts, degradation of tran- the cellular distribution of the core subunits is remarkably different 135

72 scripts and transcriptional downregulation of the cognate genes. The from that of RRP6 and DIS3 in D. melanogaster cells [35], and the 136

73 retention of faulty transcripts can occur at the gene locus or at interactions that support the association of RRP6 and the exosome 137

74 the nuclear pore. Studies in Saccharomyces cerevisiae, Drosophila core subunit RRP4 with nascent transcripts in insect chromosomes 138

75 melanogaster and mammalian cells have demonstrated that unpro- are differently affected by RNase digestions [36]. Systematic studies 139

76 cessed transcripts are retained at the site of transcription ([412]; are needed to establish the contributions of the individual exosome 140

77 see Section 5.1), but the retention of mRNPs at the nuclear pore subunits to the different functions of the exosome. 141

78 has been reported only in budding yeast. Unspliced transcripts

79 are actively retained at the nuclear side of the NPC by the NPC- 2.1. Nuclear surveillance of mRNA biogenesis by the exosome 142

80 associated proteins Mlp1/2p and Pml39p through a mechanism

81 that involves the 5 splice site [13,14]. The nuclear envelope protein A QC system should include at least two components: a sen- 143

82 Esc1p, which is important for the proper assembly of the nuclear sor that is able to identify defective mRNAs, and a degrader that 144

83 basket and for the localization of the Ulp1p SUMO protease, is also eliminates or inactivates the defective molecules before they are 145

84 required, together with Ulp1p, for the retention of defective mRNPs expressed. The nonsense-mediate mRNA decay pathway (NMD, 146

85 at the nuclear pore [15]. This suggests that desumoylation plays see Section 5.4), for example, conforms to this conguration: the 147

86 an important role in this process. There is evidence that the NPC- UPF1 protein acts as a sensor to identify PTCs and targets the PTC- 148

87 retained transcripts are degraded by Rrp6p, one of the catalytic containing transcript for degradation. However, the sensors that 149

88 subunits of the exosome ([13]; see Section 2), but little is known cooperate with the exosome in the nuclear surveillance of mRNA 150

89 about the fate of the retained transcripts. The mammalian ortholog biogenesis are not dedicated machineries. Instead, the QC carried 151

90 of Mlp1/2p, TPR, regulates the export of unspliced transcripts that out by the exosome depends on molecular interactions between the 152

91 are exported to the cytoplasm through the NXF1 pathway [16], exosome and components of the gene-expression machineries, and 153

92 which suggests that the role of the MLP proteins in the nuclear is based on the fact that correct pre-mRNP packaging and efcient 154

93 retention of unspliced mRNPs is conserved. processing counteract the destabilizing consequences of incorrect 155

94 The nuclear QC mechanisms use canonical decay enzymes for or inefcient biogenesis. This model for the activity of the exosome 156

95 the rapid and efcient degradation of faulty transcripts. These are in mRNP QC, initially proposed by Jensen et al. [37], is based on 157

96 mainly the nuclear exosome, with its ability for 3 5 exonucleolytic several assumptions. First, the exosome (or its catalytically active 158

97 and endonucleolytic decay (see Section 2) and the 5 3 exoribonu- subunits) must be recruited to the nascent transcripts. Chromatin 159

cleases (exoRNases) XRN2/Rat1p and DXO (see Sections 4 and 5.2.). immunoprecipitation and immunolocalization experiments have 160

Please cite this article in press as: Eberle AB, Visa N. Quality control of mRNP biogenesis: Networking at the transcription site. Semin
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Fig. 1. Quality control of mRNP biogenesis. Eukaryotes have evolved sophisticated QC systems that can sense defects in the processing of the pre-mRNAs, in the assembly of
the (pre)mRNP complexes, and in the sequence of the open reading frames. The nuclear QC mechanisms degrade the faulty transcripts using different RNA decay systems.
The nuclear exosome, with its ability for 3 5 exonucleolytic and endonucleolytic decay, and the 5 3 exoRNAses XRN2/Rat1p and DXO are major players in the QC of mRNA
biogenesis in the cell nucleus. mRNA QC takes place also in the cytoplasm, where different QC mechanisms take action if ribosomes encounter difculties during translation.

161 demonstrated that this is the case (see, for example, [5,21,38]), ndings on the molecular mechanisms by which these processing- 193

162 and specic protein-protein interactions that tether the exosome coupled remodeling events are linked to the QC systems. 194

163 to transcriptionally active genes have been identied (see Section The exosome competes with the pre-mRNA processing reac- 195

164 2.2). A second assumption is that the nascent (pre)mRNPs are pro- tions, as initially suggested more than a decade ago by experiments 196

165 tected from exoribonucleolytic attack under normal circumstances in which the degradation of unspliced transcripts in S. cerevisiae 197

166 [1], and only become vulnerable if a step in gene expression devi- increased signicantly in conditions of inhibited splicing [9,44]. 198

167 ates from the correct pathway. Indeed, the 3 -end of the nascent These observations led to the proposal that newly synthesized 199

168 transcript is hidden in the Pol-II catalytic core during transcrip- pre-mRNAs are either rapidly packaged into stable, competent pre- 200

169 tion elongation [39], and is therefore safe from exoribonucleolytic mRNPs and efciently processed, or degraded by the exosome [45]. 201

170 attack. The 3 -end of the pre-mRNA can become exposed if tran- However, more recent transcriptome analyses in S. cerevisiae have 202

171 scription elongation is impaired, as may occur, for instance, in the revealed that the exosome RNases also degrade a large fraction of 203

172 case of transcriptional backtracking or premature transcription ter- error-free pre-mRNAs [46]. This observation suggests that the QC 204

173 mination, and this exposure provides the basis for the surveillance carried out by the exosome is relatively promiscuous, and it indi- 205

174 of transcription elongation. The endonucleolytic activity of DIS3 cates that a fraction of the newly made pre-mRNAs is constantly 206

175 could, in principle, generate entry sites for 3 5 exonucleolytic degraded. A QC strategy based on non-productive transcription and 207

176 degradation, but the contribution of DIS3 to co-transcriptional extensive RNA degradation may be advantageous for the homeosta- 208

177 RNA cleavage is unclear, and the assembly of nascent pre-mRNA sis of RNA precursors or for the regulation of gene transcription, and 209

178 with hnRNP proteins seems to provide efcient protection against may have an impact on the structure of the chromatin, as recently 210

179 ribonucleolytic attack (see Section 3). discussed by Schmid and Jensen [47]. 211

180 A third assumption that is relevant to the mechanism by which

181 the exosome carries out the QC of nascent transcripts is that 2.2. The recruitment of the exosome to nascent transcripts 212
182 each individual step in the processing of the pre-mRNA entails a
183 remodeling of the (pre)mRNP complex. Erroneous processing reac- The mechanisms by which the exosome is recruited to newly 213
184 tions would lead to aberrant mRNP remodeling, which would, in synthesized mRNPs are still under investigation. Chromatin immu- 214
185 turn, leave the mRNA unprotected or retained at the transcription noprecipitation (ChIP) proling in S. cerevisiae has shown that 215
186 site [37]. It is well established that the (pre)mRNPs are signi- Rrp6p is broadly distributed across the genome [48]. This is also 216
187 cantly modied as a consequence of pre-mRNA processing. The the case in animal cells, where the exosome is associated with 217
188 capped 5 -end recruits CBC, the nuclear cap-binding complex [40]. many regions of the genome through interactions that are highly 218
189 The excision of introns is accompanied by the deposition of the dynamic and that depend to a large extent on transcription. The 219
190 exon junction complexes (EJC) onto the mRNP [41,42], while the use of antibodies against individual subunits of the exosome of 220
191 cleavage and polyadenylation reactions lead to the recruitment of D. melanogaster and Chironomus tentans have shown that the exo- 221
192 polyA-binding proteins [43]. The sections below summarize recent some is associated with transcriptionally active loci on polytene 222

Please cite this article in press as: Eberle AB, Visa N. Quality control of mRNP biogenesis: Networking at the transcription site. Semin
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Fig. 2. Multiple interactions support the recruitment of the exosome to nascent pre-mRNPs. The gure summarizes protein-protein interactions that can mediate the
association of the exosome with genes transcribed by Pol-II in animal cells. Co-immunoprecipitation experiments in D. melanogaster identied SPT5 and SPT6, two transcription
elongation factors, as interaction partners for the exosome [21]. In D. melanogaster, the exosome can also be tethered to the nascent pre-mRNP through interactions with the
hnRNP proteins HRP59 [38]. In human cells, an interaction between exosome factors and the nuclear CBC is mediated by the NEXT complex, and this interaction is important
to connect the exosome to Pol-II transcripts [49]. It is not known whether the exosome is rst recruited to the gene through interactions with the transcription machinery
and subsequently transferred to the pre-mRNP, or directly recruited to the transcription unit by the proteins that interact with the nascent transcript.

223 chromosomes [21,38], that it remains bound to mRNPs after splic- differences that render the non-functional transcripts exposed and 268

224 ing, and that it accompanies the spliced mRNPs to the nuclear more susceptible to degradation. 269

225 pore [36]. ChIP experiments of individual exosome subunits have

226 shown that the exosome is present along the complete length of
227 protein-coding genes, even in intronless genes. The concentration 3. QC of pre-mRNP assembly 270

228 of exosomes is highest at the 5 -end of the genes, which suggests

229 that QC starts as soon as the synthesis of the nascent pre-mRNA Nascent pre-mRNAs become rapidly associated with a variety 271

230 is initiated [38]. Physical interactions between the exosome, Pol- of RNA-binding proteins that restrict and regulate the interactions 272

231 II and the transcription elongation factors SPT5 and SPT6 suggest between the pre-mRNA and other cell components (reviewed by 273

232 that the transcription machinery recruits the exosome to sites of [1]). The major mRNA-binding proteins, also known as hnRNP pro- 274

233 pre-mRNA synthesis, and that the exosome travels along the tran- teins, have a modular structure and contain not only domains 275

234 scribed genes bound to the transcription elongation complex [21]. that bind RNA but also specialized auxiliary domains for pro- 276

235 Moreover, the exosome can be co-immunoprecipitated with sev- teinprotein interactions [50]. The protective roles of individual 277

236 eral mRNA-binding proteins, and it can interact with the nascent mRNA-binding proteins are difcult to dissect in vivo due to the 278

237 pre-mRNP independently of the transcription machinery, as shown multiple roles of these proteins and their overlapping binding 279

238 by immuno-electron microscopy studies of RRP4 and RRP6 in the specicities. However, early studies of hnRNPs in mammalian cells 280

239 Balbiani ring pre-mRNPs of C. tentans [36,38]. The HRP59 protein of showed that hnRNP complexes are much more resistant to enzy- 281

240 D. melanogaster, also known as hnRNPM and Rump, is an abundant matic degradation than the corresponding hnRNA preparations 282

241 mRNA-binding protein that interacts physically with several exo- devoid of proteins (see, for example, [51]). Collectively, the pro- 283

242 some subunits. Depletion of HRP59 by RNA interference reduces teins bound to the nascent transcripts constitute a stable protection 284

243 the levels of RRP4 at transcribed genes, which suggests that HRP59 and provide at the same time a exible interface with the process- 285

244 is needed for the recruitment or tethering of the exosome with ing and export machineries. The bound proteins not only restrict 286

245 nascent pre-mRNPs [38]. The human hnRNPM coimmunoprecip- the access of ribonucleases to the RNA but also favor the assembly 287

246 itates with MTR4 and ZCCHC8, two components of the nuclear of competent mRNP export complexes that are efciently trans- 288

247 exosome targeting (NEXT) complex, which is involved in the degra- ported to the cytoplasm and in this way evade nuclear degradation 289

248 dation of promoter upstream transcripts (PROMPTs) [22]. This (reviewed by [52]). 290

249 suggests that the role of hnRNPM in linking the exosome to nascent Perhaps the most studied case of the QC of mRNP assembly con- 291

250 transcripts is conserved in mammals. cerns the THO complex. The THO proteins are hnRNP-like proteins 292

251 The human NEXT complex also interacts with CBP80, the large that function in the cotranscriptional assembly of pre-mRNPs in 293

252 subunit of the nuclear cap-binding complex (CBC), and links the S. cerevisiae [53]. Aberrant pre-mRNPs synthesized in yeast strains 294

253 exosome to the 5 -cap [49]. This interaction promotes early tran- that carry mutations in the THO subunits are retained at the tran- 295

254 scription termination of PROMPTs and read-through RNAs, and scription site and degraded by a mechanism that requires Rrp6p 296

255 is important for the discrimination between short unstable tran- and the exosome cofactor Trf4p [54,55]. 297

256 scripts and functional pre-mRNAs. However, its signicance in the

257 surveillance of protein-coding transcripts is unclear.
258 The proteins that mediate the association of the exosome with 4. QC of 5 -end capping 298

259 transcribed genes, either components of the transcription machin-

260 ery or mRNA-binding proteins (Fig. 2), are not linked to defects in The 5 end of the pre-mRNA is capped by the addition of a 7- 299

261 gene expression and are present in all the transcription units. This methylguanosine (m7 G) linked to the rst encoded nucleotide via 300

262 implies that the exosome is recruited to newly synthesized mRNAs a 5 5 triphosphate linkage. This reaction occurs on all the tran- 301

263 independently of their quality and processing status, and that the scripts synthesized by Pol-II shortly after the start of transcription 302

264 selective degradation of unspliced or defective transcripts is not [56,57]. The nuclear cap-binding complex (CBC) recognizes the 303

265 achieved by the selective recruitment of the exosome to aberrant m7 G and protects the 5 end of the pre-mRNA from degradation 304

266 mRNAs. Instead, all transcripts seem to coexist with the exosome, [40,58]. Uncapped or incorrectly capped pre-mRNAs fail to recruit 305

267 and the QC mechanisms must thus rely on kinetic or structural the CBC and are exposed to enzymatic attack. 306

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307 The XRN2 protein, and its ortholog in yeast Rat1p, are the major 5.1. Retention at the transcription site 370

308 5 3 exoRNases in the cell nucleus [59]. XRN2/Rat1p functions in

309 the termination of transcription by Pol-II, and is responsible for the Splicing mutants of the human -globin gene have been used as 371

310 degradation of the downstream RNA products that are generated reporters to analyze the QC of pre-mRNA splicing in animal cells, 372

311 by the cleavage of the pre-mRNAs at the polyadenylation site ([60], and early studies by Custodio et al. [4] showed that mutant -globin 373

312 see Section 6). transcripts with retained introns or with defective 3 ends were 374

313 In S. cerevisiae, Rat1p acts together with the pyrophosphohy- retained at the transcription site. Impaired 3 -end processing in S. 375

314 drolase Rai1p and the decapping enzyme Dox1p. Rai1p converts cerevisiae also results in nuclear retention of transcripts near tran- 376

315 uncapped pre-mRNA ends into monophosphorylated 5 ends that scription sites [10], which supports the idea that the retention of 377

316 can be readily degraded by Rat1p [61]. Pre-mRNAs with methylated aberrant transcripts is a conserved phenomenon. The retention of 378

317 or unmethylated caps can also be decapped by Rai1p and Dox1p, 3 -end defective transcripts at the sites of transcription requires the 379

318 and subsequently degraded by Rat1p [62]. Thus, these proteins con- exosome complex, and in particular the exoribonucleolytic activity 380

319 stitute an effective QC system that degrades transcripts that lack provided by Rrp6p [9,54,68]. RRP6 is also needed for the retention of 381

320 properly protected 5 ends. A similar mechanism exists in mam- -globin transcripts that are defective in splicing in D. melanogaster 382

321 malian cells. The DOX1 protein, which is the mammalian ortholog and human cells [5,8]. 383

322 of Rai1p and Dox1, is a multifunctional enzyme with a 5 pyrophos- Two important questions that remain are the nature of the 384

323 phohydrolase activity, a decapping activity, and a 5 3 exoRNase mechanisms by which the nascent transcripts are tethered to the 385

324 activity [63]. It has not been formally shown that DXO is recruited chromatin template, and the relationships between this tether- 386

325 to transcribed genes in vivo, but DXO acts preferentially on pre- ing and splicing. It has been proposed that several mechanisms of 387

326 mRNA, not mRNA, which supports an early action [63]. Moreover, transcript retention take place in S. cerevisiae, and the proposed 388

327 capped RNAs associated with cap-binding proteins are protected mechanisms have recently been reviewed by Schmid and Jensen 389

328 from degradation by DXO, which also supports the proposal that [47]. Non-adenylated transcripts accumulate at the transcription 390

329 DXO targets defectively capped RNAs that fail to recruit the CBC site, which suggests that the efcient release of transcripts involves 391

330 [63]. a functional polyA tail [7]. Several mechanisms have been proposed 392

331 ChIP-Seq experiments have revealed that the human XRN2 in which the exosome, or Rrp6p, participates in the polyadenylation 393

332 localizes near to transcription start sites, together with the decap- of nascent transcripts or in the assembly of polyA-binding proteins 394

333 ping enzymes EDC3, DCP1a and DCP2 [64]. The extent to which on the polyA tail [69]. The association of transcribed genes and their 395

334 the mammalian XRN2 contributes to the QC of 5 capping is, how- transcripts with the nuclear pore has been proposed as an alterna- 396

335 ever, unclear. There is evidence that XRN2 plays an important role tive mechanism for the nuclear retention of transcripts in yeast 397

336 in transcription regulation. Depletion of either XRN2 or decapping [70]. 398

337 enzymes causes transcription elongation defects, and it has been The transcription machinery plays a major role in the reten- 399

338 proposed that these enzymes act near the transcription start sites to tion of defective transcripts in mammalian cells. Experiments based 400

339 induce premature transcription termination and limit bidirectional on in vitro transcription systems coupled to pre-mRNA processing 401

340 elongation by Pol-II [64]. XRN2, together with the microprocessor suggest that the nascent pre-mRNPs are bound to the Pol-II com- 402

341 and the exosome RNase RRP6, induces Pol-II pausing and prema- plex during transcription elongation through interactions that are 403

342 ture transcription termination in a subset of human genes, which released only after the splicing and polyadenylation of the tran- 404

343 suggests that these proteins participate in a mechanism that reg- script have taken place [71]. The role of Pol-II in the retention 405

344 ulates transcription elongation by Pol-II [65]. A recent study has of unprocessed transcripts has been further supported by exper- 406

345 suggested that the yeast Rat1p is also involved in transcription iments with deciently processed -globin pre-mRNAs that are 407

346 regulation [66]. stalled at the 3 end of the gene in association with Pol-II through a 408

mechanism that requires RRP6 [5]. Failure to splice the downstream 409

intron of the human -globin pre-mRNA inhibits the cleavage and 410

347 5. QC of splicing polyadenylation of the transcript [72], and this makes it difcult 411

to discern whether the RRP6-dependent stalling of Pol-II in the 412

348 Pre-mRNA splicing is a potential source of errors in the struc- -globin splicing mutants is due to splicing or 3 -end processing 413

349 ture and sequence of mRNAs, and eukaryotic cells have evolved QC defects. A more direct link between splicing and Pol-II was pro- 414

350 responses to deal with unspliced or incorrectly spliced pre-mRNAs. vided by the study of the dynamics of -globin pre-mRNA synthesis 415

351 Some of the responses appear to be conserved, but some differences and splicing using in vivo imaging techniques. This study revealed 416

352 are apparent between yeast and metazoans. Three main responses that the assembly of the spliceosome on the pre-mRNA is cou- 417

353 have been described by which eukaryotic cells deal with splicing pled to transcription termination at the 3 -end of the gene, and this 418

354 defects: the pre-mRNAs can be retained in the nucleus, the pre- coupling provides a frame for proofreading and surveillance [73]. 419

355 mRNAs can be degraded, or the transcription of genes that produce The QC of splicing also relies on the recruitment of mRNA export 420

356 defective transcripts can be shut down. These different responses factors to the nascent pre-mRNP, as shown by recent studies on 421

357 have been observed in yeast and animal cells, but it is unclear how the QC of pre-mRNA splicing in S. cerevisiae. The TRAMP com- 422

358 defective pre-RNAs are recognized and how the different mecha- plex is cotranscriptionally recruited to nascent pre-mRNAs and is 423

359 nisms are coordinated. particularly enriched at intronic sequences [74]. The SR proteins 424

360 Both the composition of the (pre)mRNP and its nucleotide Gbp2p and Hrb1p are needed for the stable binding of TRAMP to 425

361 sequence are modied during the splicing reaction, and intron spliceosome-bound transcripts [75]. Moreover, Gbp2p and Hrb1p 426

362 removal affects downstream steps of gene expression. More- also play a role in the recruitment of Mex67p, a major mRNA export 427

363 over, splicing occurs to a large extent cotranscriptionally [67], factor, to the spliced mRNAs [75]. These studies identify TRAMP as 428

364 which links the surveillance of the splicing process to the tran- an important factor for pre-mRNA splicing surveillance in yeast, 429

365 scription machinery. The complexities of the splicing process and conrm the role of the exosome in this process. 430

366 and of the scenario in which it takes place have made it The EJC is assembled onto the transcripts cotranscriptionally 431

367 difcult to examine the direct consequences of defective splic- during splicing and mediates the recruitment of the export fac- 432

368 ing separately from the effects of other aberrant processing tor NXF1 to the mRNP [76], thus establishing a functional link 433

369 events. between splicing and export to the cytoplasm. In mammalian cells, 434

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435 NXF1 does not accumulate on nascent transcripts, which suggests 5.3. Splicing and transcription downregulation 498

436 that the recruitment of the mRNA export factors is rapidly fol-
437 lowed by the release of the mRNP from the sites of transcription Feedback mechanisms reduce the transcription rates of genes 499

438 [77]. Although the EJC is important for the QC of splicing in the in response to pre-mRNA splicing defects (reviewed by [85]). 500

439 context of the mammalian NMD pathway, its role in the release Relatively well understood mechanisms of splicing-coupled tran- 501

440 of processed transcripts from the sites of transcription remains scriptional repression have been reported in Cryptococcus and in 502

441 unclear. ssion yeast, where stalled splicing intermediates recruit the RNAi 503

442 From a QC point of view, the main effect of the retention of machinery and induce RNAi-dependent heterochromatin forma- 504

443 unspliced or improperly spliced transcripts at the transcription site tion at the gene [86,87]. The RNAi machinery does not seem to 505

444 may be to prevent the export and translation of defective mRNPs. play the same role in animal cells, where different mechanisms 506

445 Retention may also provide extra time for inefcient processing of transcriptional downregulation have been proposed. As men- 507

446 reactions to occur cotranscriptionally. Indeed, studies in S. cere- tioned above, pre-mRNA processing and transcription termination 508

447 visiae have revealed that the retention of the transcripts per se are interconnected events, and defective splicing has been corre- 509

448 does not compromise their functionality, as transcripts that are rst lated with the accumulation of stalled polymerases at the 3 end 510

449 retained at the transcription site and subsequently released into the of the gene [73]. The stalled Pol-II may impair efcient recycling 511

450 cytoplasm are translationally competent [78]. Retained transcripts of the components of the transcription machinery, and thus reduce 512

451 can also be degraded, as discussed below. transcription initiation rates. An alternative proposal that is gaining 513

support is that splicing exerts a positive regulation on transcription, 514

and that ineffective splicing interrupts this positive feedback loop. 515

452 5.2. Degradation of unspliced transcripts In favor of this hypothesis, the presence of a functional 5 splice 516

site enhances the recruitment of transcription initiation factors to 517

453 The exosome is the major RNase that acts in the QC of the promoters of reporter genes expressed in human cells [88], and 518

454 pre-mRNA processing in S. cerevisiae and is responsible for the genome-wide ChIP-Seq results suggest that the rst 5 splice site of 519

455 degradation of aberrant (pre)mRNAs that accumulate in cells that a gene acts as a position-dependent enhancer that promotes tran- 520

456 are defective in (pre)mRNP assembly, processing or export, as scription initiation through a mechanism that involves increased 521

457 recently reviewed by Schmid and Jensen [47]. In mammalian H3K4me3 and H3K9ac modications [89]. Transcriptional silencing 522

458 cells, the exosome is involved also in the nuclear degradation of has also been reported in cells of D. melanogaster, where Pol-II occu- 523

459 (pre)mRNAs. Studies by West and Proudfoot on reporter -globin pancy is signicantly reduced at the promoter of a mutant -globin 524

460 genes showed that the exosome subunit RRP6 (also known as gene reporter [8]. The feedback mechanism is less well understood 525

461 EXOSC10 or PMScl100 in humans) degrades splicing-defective and in this case, but it involves also modications in histone tails and 526

462 termination-defective transcripts [79,80]. In cases of inefcient was only observed when the gene is expressed at high initiation 527

463 transcription termination, the degradation takes place after cleav- rates [8]. It is possible that splicing favors a rate-limiting event, such 528

464 age of the pre-mRNA at the polyA site, which generates an exposed as the assembly of the transcription pre-initiation complex, that can 529

465 3 end [80]. be overcome if the rate of transcription initiation is relatively low. 530

466 In mammalian cells, however, also XRN2 plays an important

467 role in the cotranscriptional degradation of splicing-defective tran- 5.4. Splicing and nonsense mutations 531

468 scripts, not only in reporter -globin systems but also when the
469 splicing of endogenous transcripts is inhibited with spliceostatin Defective splicing often results in transcripts with retained 532

470 A (SSA) [79]. How the 5 end that is required for XRN2 decay is introns that contain premature translation termination codons 533

471 generated is not totally understood. The decapping factor DCP2 (PTCs, also known as nonsense mutations). These improperly pro- 534

472 was detected by ChIP at some SSA-affected loci and may provide cessed transcripts can be exported to the cytoplasm where they can 535

473 a monophosphorylated 5 end [79]. Alternative proposed mech- engage in translation and become exposed to NMD, a cytoplasmic 536

474 anisms for the generation of free 5 ends include the processing QC mechanism by which transcripts that contain PTCs are recog- 537

475 of microRNA-containing introns [81], the endonucleolytic cleav- nized and rapidly degraded (reviewed in [9092]). Current models 538

476 age by RRP44/DIS3 [27], and the resolution of R-loops by RNase H suggest that NMD is initiated by the binding of the ATP-dependent 539

477 [82]. RNA helicase UPF1 and the serinethreonine kinase SMG1 to the 540

478 The nuclear retention and degradation of defective transcripts ribosome stalled at a PTC, probably through an interaction with the 541

479 appear to be efcient QC responses in yeast cells, but they are eukaryotic release factors eRF1 and eRF3 [93,94]. A key step in PTC 542

480 less stringent in animal cells, where the discrimination between recognition is the phosphorylation of UPF1 by SMG1, for which the 543

481 processed and unprocessed is probably less straightforward, presence of UPF2 and UPF3, possibly bound to a downstream EJC 544

482 given that alternative pre-mRNA processing events are much more on the mRNP, is crucial [94,95]. SMG5, SMG6 and/or SMG7 subse- 545

483 prevalent in animal cells. Studies of viral infection cycles in mam- quently bind to the phosphorylated UPF1 through their 14-3-3-like 546

484 malian cells have provided examples of situations in which both domain, which leads to efcient decay of the transcripts [9699]. 547

485 spliced and unspliced RNAs are exported to the cytoplasm [83]. Degradation of the NMD substrates is intimately connected with 548

486 Cellular transcripts that are not properly spliced are also exported dephosphorylation of UPF1, along different, partially redundant, 549

487 to some extent, as can be inferred from the fact that abnormally degradation pathways in mammalian cells (reviewed in [100]). 550

488 spliced globin transcripts are sensitive to NMD in humans (see, for One such pathway relies on the endonucleolytic activity of SMG6 551

489 example, [84]). Signicant export of intron-containing mRNAs to [101103], whereas others involve the exosome or the decapping 552

490 the cytoplasm occurs in cells of D. melanogaster after splicing has activity of DCP1A [104108]. 553

491 been inhibited by the depletion of splicing factors (Eberle and Visa, NMD substrates are very diverse and include not only mRNAs 554

492 unpublished). These observations underscore the fact that the indi- with PTCs but also transcripts with introns in the 3 -UTR and tran- 555

493 vidual mechanisms of the QC of splicing may not be sufcient to scripts with long 3 -UTRs in which the physiological stop codon 556

494 ensure the silencing of all defectively spliced transcripts in animal is recognized as premature. Large-scale analysis in human cells 557

495 cells, where the accuracy of the splicing outcome probably relies on revealed that about one third of all alternatively spliced tran- 558

496 the combined action of several QC responses that act at different scripts contain a PTC and are therefore potential targets for NMD 559

497 levels. [109]. This fact, together with deep sequencing studies of the 560

Please cite this article in press as: Eberle AB, Visa N. Quality control of mRNP biogenesis: Networking at the transcription site. Semin
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561 transcriptome showing that 9294% of all human multi-exon genes protein Pab2p targets RNAs for degradation by the nuclear exosome 624

562 undergo alternative splicing [110,111], indicates that nonsense [128,129]. Thus, the two nuclear polyA binding proteins of S. pombe, 625

563 mRNAs are very abundant in higher eukaryotes. Pab2 and Nab2, play opposite roles in regulating the stability of the 626

564 Apart from inducing transcript degradation via the NMD path- transcripts [130]. 627

565 way in the cytoplasm, the presence of nonsense mutations can Impaired 3 end processing can also feed back to transcrip- 628

566 feed back to the nucleus and inuence transcription and pre-mRNA tion activity. A study by the Jensen lab reported that transcription 629

567 processing. Introduction of a PTC into reporter genes can inu- initiation rates decreased due to point mutations in the PAS of 630

568 ence the splicing pattern of the pre-mRNA through a mechanism reporter genes that were stably integrated as single copies in the 631

569 termed nonsense-associated altered splicing (NAS) [112114]. Using genome of mammalian cells [131]. In addition, the depletion of 632

570 uorescent in situ hybridization, Mhlemann et al. found that a PTC- the 3 end-processing and transcription-termination factor PCF11 633

571 containing reporter transcript accumulated in its unspliced form at had a similar effect on the transcription initiation of endogenous 634

572 the transcription site through a mechanism that depended on the transcripts. These Pol-II complexes fail to terminate properly, sug- 635

573 reading frame [11]. A more recent study revealed that the accumu- gesting that defects in 3 end processing interferes with the release 636

574 lation at the site of transcription depends on UPF1 and SMG6, two and recycling of initiation and elongation factors [131]. This com- 637

575 NMD factors that are associated with transcription sites [6]. Deple- munication between the beginning and the end of a gene could 638

576 tion of UPF1 alters the rate of Pol-II transcription elongation [6], be sustained by gene loops [132]. Lykke-Andersen has discussed 639

577 which suggests that UPF1 is involved in transcription regulation. several models that explain how the processing of the 3 end can 640

578 In support of this idea, PTC has a feedback effect on the transcrip- stimulate transcription initiation [133]. 641

579 tion of Ig- and Ig- reporter genes [115]. This phenomenon of Recent studies have revealed the existence of PASs located 642

580 nonsense-mediated transcriptional gene silencing was accompanied upstream of gene promoters in vertebrate genomes. These 643

581 by chromatin remodeling and changes in the post-translational upstream PAS elements are abundant in antisense PROMPTs, and 644

582 modications of histone tails [115]. Furthermore, translation of target the transcripts for degradation instead of supporting effec- 645

583 the PTC-containing reporter transcript and UPF1 were required tive 3 end processing. In this way, the high density of PASs in the 646

584 for transcriptional downregulation [116]. However, the molecular upstream antisense regions contributes to impose promoter direc- 647

585 mechanism of this transcriptional feedback and its physiological tionality, and limits pervasive transcription by promoting early 648

586 signicance are unknown [117]. termination and RNA degradation in upstream antisense regions 649

[134,135]. A question that remains to be answered is why the 650

upstream PASs fail to engage in proper processing. 651

587 6. QC of 3 end formation

588 The maturation of the 3 end of most protein-coding transcripts 7. Integration of mRNA decay systems at the site of 652

589 involves an endonucleolytic cleavage of the transcript, followed transcription 653

590 by the addition of a polyA tail to the 3 end that is generated

591 by the cleavage. The polyA tail is synthesized by dedicated polyA The exosome, the enzymes involved in 5 3 mRNA decay, and 654

592 polymerases and is crucial for nuclear export, stability and the ef- some NMD factors are connected to each other through multiple 655

593 cient translation of the mRNA (reviewed in [118]). Cleavage and proteinprotein interactions, which provides the opportunity for a 656

594 polyadenylation need several cis-acting elements on the transcript: coordinated mode of action [136]. The picture that emerges from 657

595 a conserved polyadenylation signal (PAS) (A[A/U]UAAA or close many studies on the mechanisms of the QC of mRNA biogenesis is 658

596 variants of this), a cleavage site located about 1030 nt downstream that these major mRNA decay systems coexist at the sites of tran- 659

597 of the PAS, and GU- or U-rich downstream sequence elements. scription, in close connection either with the nascent pre-mRNPs 660

598 These elements are essential for the proper positioning of the or with the transcription machinery. The interactions detected in 661

599 numerous trans-acting factors that constitute the 3 end-processing yeast two-hybrid assays, co-immunoprecipitation, and ChIP exper- 662

600 machinery (reviewed in [119]). iments suggest that the transcribed genes integrate multiple mRNA 663

601 All pre-mRNA processing steps, including 3 end formation, decay systems into a complex QC network (Fig. 3). 664

602 are tightly coupled to transcription and to each other (reviewed Many pre-mRNA processing reactions take place cotranscrip- 665

603 in [120123]). Defects in the processing of the 3 end can lead tionally and need to be monitored, and other types of RNA, apart 666

604 to retention of the transcripts at the site of transcription in an from the nascent pre-mRNAs, concur at the transcription sites. 667

605 exosome-dependent manner. This is not surprising, since the addi- These can be non-coding RNAs that result from bidirectional or 668

606 tion of the polyA tail plays an important role in the release of the pervasive transcription initiation events [137], abortive transcripts 669

607 mRNA from the transcription complex [71]. The polyA tail is also synthesized during promoter-proximal pausing of Pol-II [138], or 670

608 central for RNA stability, and transcripts with improper 3 ends are short regulatory RNAs that are important players in the regulation 671

609 degraded by the exosome or by XRN2 [68,79,124]. Mutation of the of gene expression in some organisms (see, for example, [139]). All 672

610 PAS or impaired cleavage and polyadenylation results not only in these different RNA species must be managed, either regulated or 673

611 defects in 3 end processing, but also in a low steady-state level of degraded, and it is thus not surprising to nd RNA-decay enzymes 674

612 mRNAs [79,80,124126]. associated with the transcribed genes. 675

613 Interestingly, the catalytic exosome subunit Rrp6p interacts The concentration of RNases at the sites of transcription con- 676

614 physically in S. cerevisiae with the polyA polymerase Pap1p and stitutes in itself a surveillance system that imposes demands on 677

615 with a polyA-binding protein called Npl3p [127]. Rrp6p controls the the structure of the nascent pre-mRNPs and on the kinetics of 678

616 length of the polyA tail by counteracting the extension of the polyA the processing and export processes. Moreover, recruiting the QC 679

617 tail by the non-canonical polyA-polymerase Trf4p, which is a com- machineries to the sites of transcription provides a frame in which 680

618 ponent of the TRAMP complex [69]. Furthermore, Rrp6p controls the QC responses can feed back to the transcription machinery and 681

619 the stability of the transcripts by inuencing the composition of the to the chromatin structure, to turn down pre-mRNA synthesis in 682

620 proteins that bind to the polyA tail. Rrp6p can interact physically response to gene-expression defects. 683

621 with another polyA-binding protein, Nab2p, and displace it from Many questions remain to be answered concerning the co- 684

622 the polyA tails, which leads to RNA degradation [69]. Similar stud- transcriptional QC. To what extent are the different mRNA decay 685

623 ies in Schizosaccharomyces pombe showed that the polyA-binding systems redundant? Do they coexist throughout the genome or are 686

Please cite this article in press as: Eberle AB, Visa N. Quality control of mRNP biogenesis: Networking at the transcription site. Semin
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Fig. 3. The human mRNA degradation systems interact with each other and with the transcribed genes. The gure summarizes proteinprotein and ChIP interactions detected
in human cells. EXOSC10 is also known as RRP6. The proteinprotein interactions (red lines) were detected by yeast two-hybrid assays [136] and co-immunoprecipitation
[140]. The ChIP results (blue lines) were reported by [5,6,64,65,79].

687 there mechanisms to target certain enzymes to specic subsets of [9] Hilleren P, McCarthy T, Rosbash M, Parker R, Jensen TH. Quality con- 725

688 genes? Are the different decay systems coordinated and, if so, how? trol of mRNA 3 -end processing is linked to the nuclear exosome. Nature 726
2001;413:53842. 727
689 Much of the available knowledge on the mechanisms of mRNP QC [10] Jensen TH, Patricio K, McCarthy T, Rosbash M. A block to mRNA nuclear export 728
690 has come from studies of reporter systems or of a reduced number in S. cerevisiae leads to hyperadenylation of transcripts that accumulate at the 729
691 of genes that can hardly provide a representative overview of the QC site of transcription. Mol Cell 2001;7:88798. 730
[11] Muhlemann O, Mock-Casagrande CS, Wang J, Li S, Custodio N, Carmo-Fonseca 731
692 mechanisms. The detailed and systematic study of the different QC M, et al. Precursor RNAs harboring nonsense codons accumulate near the site 732
693 pathways genomewide in conditions that are closer to physiolog- of transcription. Mol Cell 2001;8:3343. 733
694 ical conditions will shed light on important questions concerning [12] Thomsen R, Libri D, Boulay J, Rosbash M, Jensen TH. Localization of nuclear 734
retained mRNAs in Saccharomyces cerevisiae. RNA 2003;9:104957. 735
695 redundancy, coordination and gene specicity.
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Mlp1. Cell 2004;116:6373. 738

696 Acknowledgments [14] Palancade B, Zuccolo M, Loeillet S, Nicolas A, Doye V. Pml39, a 739
novel protein of the nuclear periphery required for nuclear reten- 740
tion of improper messenger ribonucleoparticles. Mol Biol Cell 2005;16: 741
697 We thank Marie hman for critical reading of the manuscript 525868. 742
698 and George Farrants for English editing. Our research is funded by [15] Lewis A, Felberbaum R, Hochstrasser M. A nuclear envelope protein linking 743
nuclear pore basket assembly, SUMO protease regulation, and mRNA surveil- 744
Q2 The Swedish Research Council and The Swedish Cancer Society. A.B.
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700 Eberle has been the recipient of fellowships from the Wenner-Gren [16] Coyle JH, Bor YC, Rekosh D, Hammarskjold ML. The Tpr protein regulates 746
701 Foundation and the Swiss National Science Foundation. export of mRNAs with retained introns that trafc through the Nxf1 pathway. 747
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