La deletion from mouse brain alters pre-tRNA metabolism and accumulation of pre-5.8S rRNA, with

60 Pages·2017·1.37 MB·English

MCB Accepted Manuscript Posted Online 21 February 2017
Mol. Cell. Biol. doi:10.1128/MCB.00588-16
Copyright © 2017 American Society for Microbiology. All Rights Reserved.
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3 La deletion from mouse brain alters pre-tRNA metabolism and accumulation of pre-
4 5.8S rRNA, with neuron death and reactive astrocytosis
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6 Running title: Faulty tRNA and rRNA processing and neuron loss after La deletion D
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7 n
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8 Nathan H. Blewett1, James R. Iben1, Sergei Gaidamakov1 and Richard J. Maraia2# ad
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10 1Intramural Research Program, Eunice Kennedy Shriver National Institute of Child Health and m

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11 Human Development, National Institutes of Health tp
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12 2Commisioned Corps, U.S. Public Health Service, Rockville, Maryland
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14 #To whom correspondence should be directed at: .
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15 6 Center Drive, /

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16 Bld 6A, Rm 2A02, A
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17 Bethesda, MD 20892 il
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18 E-mail: [email protected] 0
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20 Word count: Materials and Methods: 1462 u
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21 Word count: Introduction, Results, and Discussion: 6492 t
22 Characters w/o spaces: Abstract, Intro, Results, Discussion & Fig Legends: 40706
23 Conflict of Interest: The authors declare no competing financial interests.
24
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25 ABSTRACT
26 Human La antigen (Sjögren's syndrome antigen B, SSB) is an abundant multifunctional RNA-
27 binding protein. In the nucleoplasm, La binds to and protects from 3' exonucleases, the ends
28 of precursor-tRNAs and other transcripts synthesized by RNA polymerase III, and facilitates
29 their maturation, while a nucleolar isoform has been implicated in rRNA biogenesis by
30 multiple independent lines of evidence. We showed earlier that conditional La knockout (La D
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31 cKO) from mouse cortex neurons results in defective tRNA processing although pathway(s) n
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32 involved in neuronal loss thereafter was unknown. Here we demonstrate La is stably d
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33 associated with a spliced pre-tRNA intermediate. Microscopic evidence of aberrant nuclear f
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34 accumulation of 5.8S rRNA in La cKO is supported by ten-fold increase in a pre-5.8S rRNA
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35 intermediate. To identify pathways involved in subsequent neurodegeneration and loss of
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36 brain mass in the cKO cortex we employed mRNA-Seq, immunohistochemistry and other c
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37 approaches. This revealed robust enrichment of immune and astrocyte reactivity in La cKO s
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38 cortex. Immunohistochemistry including temporal analyses demonstrated neurodegeneration r
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39 followed by astrocyte invasion associated with immune response and decreasing cKO cortex
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40 size over time. Thus, deletion of La from post-mitotic neurons results in defective pre-tRNA p
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41 and pre-rRNA processing, and progressive neurodegeneration with loss of cortical brain mass. ,

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42 1
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43 INTRODUCTION
44 La was described as a component of ribonucleoprotein particles (RNPs) targeted by
45 autoantibodies in patients suffering from systemic lupus erythematosus and Sjögren's
46 syndrome (76) and later found in all free-living eukaryotes (18). La is an abundant RNA-
47 binding protein that recognizes UUU-3'-OH that results from transcription termination by
48 RNA polymerase III (RNAP III) (114, 124, 129). This conserved activity functions to protect D
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49 nascent RNAs from 3' exonucleases including the nuclear exosome-Rrp6, and facilitate the n
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50 ordered processing and maturation of these transcripts, the more abundant of which are the d
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51 precursor-tRNAs (12, 14, 25, 28, 59, 61, 64, reviewed in 83, 150, 153). f
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52 In addition to 3' end protection, La also exhibits RNA chaperone activity, which serves to
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53 prevent misfolding of those pre-tRNA sequences with propensity to form alternate structures
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54 (25, 71, 150, 151). The RNA chaperone activity works with the 3' protection activity to assist c
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55 structurally-challenged pre-tRNAs that would otherwise succumb to nuclear surveillance via s
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56 exosome-Rrp6-mediated decay (13, 28, 29, 59, 153). r
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57 La is nonessential in yeast (14, 150), but is essential during D. melanogaster and M.
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58 musculus development (8, 105), suggesting that its role in RNAP III transcript maturation is p
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59 required in metazoa. By this reasoning, metazoa may contain an essential La-dependent ,

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60 RNAP III transcript(s). La proteins also interact with viral and cellular mRNAs, including 5' 1
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61 terminal oligopyrimidine (TOP) mRNAs encoding ribosome subunits, to affect their y

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62 translation (1, 19, 24, 30, 31, 63, 90, 111, 121, 126). Furthermore, metazoan La proteins e
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63 contain an atypical RNA recognition motif (RRM) (102, 152) not found in the yeast
64 homologs, that likely extends its repertoire of RNA substrates and convey function beyond 3'
65 end protection (87). Finally, as detailed below, several lines of evidence implicate La in the
66 biogenesis of large rRNA. Thus, an alternative possibility to account for the disparity in
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67 essentiality in metazoa but not yeast that is independent of RNAP III, would be that La is
68 required in higher eukaryotes for the proper metabolism or function of an RNAP II transcript,
69 and/or a pre-rRNA species produced by RNAP I.
70 La was estimated at 2x107 molecules per cell (49), comprised of multiple phosphorylated
71 isoforms found in the nucleoplasm, nucleolus and cytosol, differentially associated with
72 nascent RNAP III transcripts or mRNAs (19, 62-64, 123, 128). In addition to the major D
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73 phosphorylation site, Ser-366 representing ~85% of La, phosphorylation by AKT can n
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74 modulate its nucleo-cytoplasmic distribution and the translation of specific mRNAs in mouse d
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75 glial progenitor cells (20, 72). f
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76 La Ser-366 phosphoprotein is nucleoplasmic, associated with nascent pre-tRNAs (63).
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77 The Ser-366 nonphospho isoform comprises ~15% of La or about 3x106 molecules/cell (40). :
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78 Ser-366 nonphospho-La is most concentrated in the dense fibrillar component of the nucleolus b
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79 localized with newly synthesized RNAP I transcripts where it physically interacts with m
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80 nucleolin (62, 63), a pre-rRNA associated protein (2, 47, 48). La was shown to interact with r
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81 nucleolin by fluorescence resonance energy transfer (FRET) and independently by yeast two n

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82 hybrid assays (62). It was proposed that nucleolin serves as a chaperone for pre-rRNA (2, also r
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83 see 46). Because La exhibits RNA chaperone activity (13, reviewed in 14, 25, 59) it is ,
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84 tempting to speculate that it may assist nucleolin in this capacity. Still, evidence that La 9

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85 functionally impacts rRNA biogenesis in a model system has been lacking.
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86 We previously reported a conditional La knockout (La cKO) gene deletion in mouse s
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87 cortex using CaMKIIα-promoter Cre recombinase which is activated in the hippocampus 3-5
88 weeks after birth and later more extensively in the cortex ((45), and refs therein). La cKO
89 brains are normal until ~6 weeks then fail to gain mass at the normal rate and by 12-13 weeks
Tyr
90 begin to lose cortical mass relative to controls (45). Defective processing of pre-tRNA was
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91 documented. Specifically, La cKO cortex exhibited substantial decrease of a 3' trailer-
Tyr Tyr
92 containing, spliced pre-tRNA but not the nascent 3' trailer-containing pre-tRNA (45),
93 surprising because both are expected to be similarly protected by La and labile in its absence
94 (64, 86, 153). In any case, the data are consistent with reports that indicate neuronal sensitivity
95 to faulty tRNA biogenesis (52, 65, 130) (reviewed in 3).
96 Here, we document accumulation of what appears to be a normal pre-5.8S rRNA D
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97 processing intermediate in La cKO cortex. We show that the 3' trailer-containing, spliced pre- n
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98 tRNA is associated with La in WT cortex providing insight into its loss in La cKO cortex. e
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99 As expected, the pre-5.8S rRNA does not appear to be associated with La, suggesting that ro
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100 effects of La deletion on its accumulation are indirect in La cKO cortex, speculatively due to h
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101 competitive inhibition of 3' exonucleases required for 5.8S maturation by nascent pre-tRNAs //
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102 and other RNAP III transcripts in the absence of 3' binding by La. mRNA-seq done on 16- b
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103 week cortex revealed robust immune activation in La cKO that coincides with prolific m
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104 astrocytosis. Temporal analyses indicate onset of astrocyte invasion and neuron loss coincide, g
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105 beginning at 11-13 weeks. The high degree of mRNA changes due to immune cell reactivity
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106 overshadow direct effects of La deletion on specific mRNAs, especially since cKO cells are ril
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107 lost from the population, although numerous TOP mRNAs were significantly decreased. The 2
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108 data indicate that La deletion from post-mitotic neurons is associated with defects in pre- 9

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109 tRNA and pre-rRNA processing, progressive neuron damage and death, immune response g
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110 with astrocytosis, and diminution of brain mass. The La cKO model shares features with s
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111 some chronic neurodegenerative diseases involving immune activation and astrocytosis.
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113 MATERIALS AND METHODS
114 All mouse housing, husbandry, handling and studies were done at the NIH under protocol
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115 ASP 10-005, approved by the IACUC of NICHD.
116 RNA isolation. 2 wild type (WT) females and 2 La cKO mice (1 male, 1 female) were
117 sacrificed by cervical dislocation, brains removed and washed three times in 1XHBSS + 40
118 U/mL RNasin. The La cKO mice bearing PCR-verified genotypes, Lax/−, CreCaMKII, were
119 as described (45). WT mice had the La+ allele (45). Samples was handled independently to
120 produce two biological duplicates for WT and cKO. Cortices were isolated, transferred to a D
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121 glass dounce, homogenized in TriReagent, combined with 200 ul each of ddH2O and n
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122 chloroform and vortexed. After phase-extraction, RNA was precipitated with 1/10 volume 3M d
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123 NaOAc + 3 volumes EtOH. RNA was subjected to bioanalyzer analysis to ensure sample fr
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124 uniformity and high quality. RNA samples were transferred to NISC (NIH Intramural h
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125 Sequencing Center, Rockville) for polyA+ selection and Illumina sequencing. RNAs for :
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126 northern blotting were isolated the same way but from different biolgical duplicates. b
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127 RNA-seq data analysis. Each WT and LaKO sample was analyzed independently as one of m
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128 two biological duplicates. Gene Ontology analysis was performed using three independent r
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129 widely-used methods: DAVID (57), PANTHER (93, 131) and Network Ontology Analysis n

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130 (145). GO analysis was performed on mRNAs which were altered to a statistically significant r
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131 degree (p-value <.06) and found to be 2-fold increased or decreased in the La cKO cortex. ,
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132 Redundant and overly general GO terms were omitted. Terms are listed with their respective 9

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133 p-values to indicate the statistical power of each enriched term.
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134 TOP mRNA analysis: All mRNAs found to be decreased to a statistically significantly level s
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135 (26% cut-off) in La cKO relative to WT, subjected to DAVID gene ontology (GO) analysis
136 that revealed enrichment of term "ribosome" were listed in Table III.
137 qRT-PCR. Isolated RNA was DNAse treated, extracted with phenol:chloroform and
138 precipitated. cDNAs were transcribed using gene-specific primers with SuperScriptIII reverse
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139 transcriptase (Life Technologies #18080400) according to manufacturer's protocol. qPCR
140 was performed (SybrGreen PCR master mix Applied Biosystems #4309155) with 2 ul of each
141 cDNA reaction, using an ABI Prism 7000 sequence detection system. Amplification
142 efficiency and melting curves were determined for each primer set to ensure accurate qPCR
143 results. Relative mRNA abundance was calculated via the ΔΔCt method, from the biological
144 duplicate samples using GAPDH mRNA for normalization, errors bars indicate standard D
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145 deviation. The qRT-PCR RNA samples were prepared from different mice than the samples n
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146 that the RNA-seq was performed on. d
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147 qRT-PCR Primers: C4b forward 5’-CGCCTGCCCATCTCCATC, C4b reverse: 5’- f
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148 TGGACACTCACAGCCACATC, CD74 forward: 5’-GGCTCCACCTAAAGTACTGA-CC,
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149 CD74 reverse: 5’-TACCGTTCTCGTCGCACTTG, H2-Aa forward: 5’-TTTGACC-
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150 CCCAAGGTGGACT H2-Aa reverse: 5’ GCTTGAGGAGCCTCATTGG-TA, H2-Ab c
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151 forward 5’-CAGGAGTCAGAAAGGACCTCG, H2-Ab reverse: 5’-ACTGGCAGTCA- s
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152 GGAATTCGG, IL1b forward: 5’-TGCCACCTTTTGACAGTGATG 3’, IL1b Reverse: 5’- r
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153 TGATGTGCTGCTGCGAGATT, GAPDH forward: 5’-GCTCTCAATGACAACTT-
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154 TGTCAAGCTCATTTC, GAPDH reverse: 5’-TAGGGCCTCTCTTGCTCAGTGTCCT p
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155 Northern blot analysis. 20 ug of RNA isolated from mouse cortex was denatured and ,

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156 separated in either 8%, 10% (IP) TBE-urea polyacrylamide gels (ITS2 analysis), or 1.2% 1
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157 denaturing formaldehyde agarose gels (ITS1 analysis). After electrophoresis RNA was y

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158 transferred to nylon membrane with iBlot transfer apparatus for polyacrylamide gels (Thermo- e
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159 Fisher) or by cappilary action for agarose gel, UV crosslinked and vacuum baked at 80°C.
160 Antisense oligonucleotides were 5′ labeled with 32PγATP with T4 polynucleotide kinase.
161 GAPDH 32PαUTP labeled riboprobe was produced from GAPDH PCR product using
162 MaxiScript T7 transcription kit (Thermo cat# AM1324) and hybridized overnight. Blots were
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163 pre-hybridized for 2 h in 15 ml hybridization buffer (6X SSC [1× SSC is 0.15 M NaCl plus
164 0.015 M sodium citrate], 0.5% SDS, 2X Denhardt's solution [catalog number 750018; Life
165 Technologies], 100 μg/ml S. cerevisiae total RNA) at the incubation temperature (T) [T = T
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166 (melting temperature) − 15°C, where T is equal to 16.6 log(M) + 0.41(P ) + 81.5 − P −
m gc m
167 (B/L), where M is the molar salt concentration up to a maximum of 0.5; P is the percent G+C
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168 content in the oligonucleotide DNA probe; Pm is the percentage of mismatched bases, if any; D
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169 B is 675; and L is the oligonucleotide DNA probe length]. Blots were washed 4 times in for n
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170 15 min at room temperature before being exposed to phosphorimager screen. Northern blots d
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171 were washed 3 x 10 min at RT with 2X SSC plus 0.1% SDS, then 2 x 15 minutes at 15°C f
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172 below the Ti. For additional probings, blots were stripped with 0.1× SSC, 0.1% SDS at 95°C,
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173 monitored to validate removal of radioactivity and then reprobed.
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174 Probes: ITS2 (pre-5.8S): 5’-ACCCACCGCAGCGGGTGACGCGATTGATCG (2263-2292 c
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175 of rRNA; begins 3 nt 3' of 5.8S and extends 30 nt), ITS1: 5’-CTCTCACCTCACTCCAGAC- s
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176 ACCTCGCTCCA-3, 5.8S mature: 5’-AAGTGCATTTGAAGTGTCAATGATCAATGTGT- r
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177 CCTGCAGTT, SRP RNA: 5’-CGAGTAGCTG-GGACTACAGGCGGTG, pre-U3 snoRNA: o
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178 5’-AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA, pre- p
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179 U8 snoRNA: 5’-AAGTGTGATCGCCAGGGAATCAGATAGGAGCAA, pre-tRNATyr ,

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180 intron: 5’-TACCATATTGTACTGACTACAGTCC, pre-tRNATyr 3' trailer: 5’-AAGTAGTG- 1
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181 CACGAAGTCCTTCG, tRNATyr spliced: 5’-AGCGACCTAAGGATCTACAGT. These are y

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182 to the mouse tRNA-Tyr-GTA-2-1 gene; chr5.trna45-TyrGTA (26). e
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183 RNA immunoprecipitation: 13 week old mice were euthanized by cervical dislocation,
184 brains removed and washed 3 times in 1X PBS + 40 U/mL SupeRasin. Cortex was isolated
185 and dounce homogenized in IP lysis buffer: (IPLB) 50 mM HEPES pH 7.0, 150 mM NaCl,
186 0.05% NP-40, EDTA-free protease inhibitor tablets (Roche), 1 mM EDTA, 40 U/mL
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187 SupeRasin. Extracts were centrifuged for 10 min. @ 3000 x g at 4°C, supernatant isolated
188 and clarified by centrifugation for 10 min @ 8000 x g. 8 ug of anti-mLa IgG (105) or mouse
189 IgG as control was immobilized on protein G dynabeads. Beads were pre-incubated with 250
190 ug/mL BSA, washed 3 times with 500 ul IPLB, then incubated with 1 mg extract in 1 ml
191 IPLB for 2 hr at 4°C. Supernatant was removed, and beads washed 5 times with 700 ul IPLB.
192 After final wash, beads were resuspended in 50 ul H2O, and RNA was extracted with acid D
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193 phenol:chloroform. RNA as separated on 10% polyacrylamide gel containing TBE-urea. RNA n
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194 was transferred to nylon membrane and hybridized with the indicated oligonucleotide probes. d
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195 Perfusion/fixation and brain sectioning. Mice were deeply anesthetized with isofluorane. f
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196 Transcardial perfusion was performed via gravity flow using sterile 1X PBS, followed by 4%
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197 paraformaldehyde/1X PBS. Brains were then removed and post-fixed in 4% PFA for 4 hrs.
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198 Brains were then cryoprotected in graded sucrose solutions containing (156 mM NaH PO , c
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199 107 mM NaOH, 0.01% thimerosal). Brains were placed in 4% sucrose for 4 hours, then 20% s
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200 sucrose for 12 hours, then 30% sucrose for 24 hours. 30 μm-thick floating sagittal brain r
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201 sections were made using a Leica SM2000R sliding microtome. For Neuroscience Associates
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202 brain block preparation, 24 mice were perfused as above, then post-fixed for 24 hours in 15% p
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203 sucrose, 1X PBS, 4% paraformaldehyde. Sex for wild type mice was: 11 weeks (2 females, 1 ,

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204 male), 13 weeks (1 female, 2 males), 16 weeks (2 females, 1 male), 20 weeks (2 males, 1 1
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205 female). Sex for La cKO mice was: 11 weeks (2 females, 1 male), 13 weeks (1 female, 2 y

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206 males), 16 weeks (3 females), 20 weeks (2 females, 1 male). Brains were shipped to e
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207 Neuroscience Associates (NSA, Knoxville, https://www.neuroscienceassociates.com/). NSA
208 embedded all 24 fixed brains into one gelatin block such that after sectioning each 30 um
209 section contains a slice of all 24 samples in one immobilized block section. Sections were
210 stored at -20°C in cryoprotectant solution.
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211 Tiled images of brain sections: Tiled 10X magnification images were of entire brain section
212 slices from WT and La cKO mice at 11-20 weeks. Tile stitching was performed using FIJI
213 Image J software and the stitching grid collection plugin (110). Equal pixel areas were
214 centered over brain sections, and cropped to preserve scale. Images from biological replicates
215 (not shown) are consistent with those depicted.
216 Confocal immunofluorescence. Antibodies were titrated to determine the optimal signal to D
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217 background conditions. Antigen retrieval was performed on floating sections in citrate buffer n
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218 pH 6.0 using a Pelco BioWave Pro. After antigen retrieval sections were blocked in 1X PBS, d
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219 20% normal goat serum, 0.5% BSA, 0.3% Triton X-100 for 1 hr. Antibodies were then added f
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220 at indicated concentrations and incubated with sections overnight at 4°C with shaking.
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221 Sections were washed 3 times for a total of 30 minutes, then appropriate secondary Abs were
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222 added and incubated for 1 hr at 25°C. Sections were washed 2 times for a total of 20 minutes, c
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223 then transferred to 1X PBS solution for 5 min. Sections were transferred to gelatin coated s
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224 slides and coverslips were mounted with MOWIOL. Imaging was performed with a Zeiss r
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225 LSM780 confocal microscope. NSA performed immunohistochemical staining for GFAP and
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226 Iba1 as per their standard optimized protocols; for published images of NSA staining for Iba1 p
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227 and GFAP, see (51, 158). ,

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228 Antibodies. Chicken anti-GFAP 1:5000 (ab4674). Rabbit anti-mouse La 1:5000 (anti-mLa; 1
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229 polyclonal against recombinant mouse La) (105). Goat anti-chicken Alexa647 1:1000 y

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230 (ab150175) Donkey anti-rabbit Alexa488 1:1000 (Life Technologies #A-21206) Y10b anti- e
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231 5.8S 1:2000 (Thermo-Fisher #MA5-16064) anti-gamma actin (Thermo Fisher #PA1-16890)
232 Rat anti-parvalbumin (Sigma-Aldrich # SAB2500752).
233 Nuclear size. 40X confocal images of DAPI stained WT and La cKO cortex were subjected
234 to Nuclear Morphometric Analysis (42) using the NII plugin for ImageJ (120).
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