The RNA binding protein Nab2 genetically interacts with multiple RNA exosome cofactors to regulate target RNAs Restricted; Files Only

Kinney, Christy (Spring 2022)

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RNA binding proteins play important roles in the processing and precise regulation of RNAs. Highlighting the biological importance of RNA binding proteins is the increasing number of human diseases that result from mutations in genes that encode these proteins. We recently discovered that mutations in the ZC3H14 gene, which encodes an evolutionarily conserved poly(A) tail RNA-binding protein, cause intellectual disability. The majority of studies that have provided insight into the function of ZC3H14 have exploited the budding yeast model to study the ZC3H14 orthologue, Nab2. The NAB2 gene is essential in S. cerevisiae and conditional nab2 mutants cause defects in a number of RNA processing steps. To explore the critical functions of the Nab2/ZC3H14 protein family, we performed a high-copy suppressor screen on nab2 mutant yeast cells. This screen identified genes encoding two core subunits of the RNA exosome, Rrp41 and Rrp42, as well as Nrd1 and Ski7, nuclear and cytoplasmic cofactors of the RNA exosome, respectively. We also identified nucleolar cofactor Nop8 and nuclear cofactor Mtr4 as suppressors; however, we focused this study on elucidating the genetic interactions between Nab2 and cofactors Nrd1 and Ski7. Using structure function analysis, we determined that the RNA binding function of Nrd1 is required for the suppression of growth defects in nab2 mutant cells, while the RNA exosome-interacting domain is required for Ski7-mediated suppression. In conjunction with previous data, our results support a model for RNA exosome impairment through overexpression of cofactors. We also used RNA-seq analysis to identify transcriptomic effects of overexpression of suppressors in nab2 mutant cells. We first compared the transcriptome of nab2 mutant to control cells and identified significantly affected transcripts and gene ontology categories. We next examined the effects of overexpression of suppressors in nab2 mutant cells and revealed distinct transcriptomes with broad but subtle effects on RNAs. These results are consistent with a global role of Nab2 in modulating transcript stability. This study uncovers functional interactions between the RNA exosome and Nab2 in both the nucleus and the cytoplasm.

Table of Contents

Chapter 1: General Introduction                                                                        1

1.1 The many classes of RNA                                                                               2

1.2 The budding yeast model system is used to study evolutionarily conserved

RNA processing steps                                                                                          2

1.3 RNA binding proteins play pivotal regulatory roles across species              3

1.4 RNA binding proteins in plants                                                                       3

1.5 RNA binding proteins are implicated in many human diseases                           4

1.6 ZC3H14 is implicated in a non-syndromic form of intellectual disability                   5

1.7 ZC3H14 is a highly conserved RNA binding protein                                        5

1.8 Nab2 mutants in S. cerevisiae are used to provide insight into potential

functions of ZC3H14                                                                                             6

1.9 Nab2-C437S is a cold-sensitive mutant                                                               7

1.10 Characterizing nab2-C437S using a high-copy suppressor screen                 8

1.11 RNA exosome subunits and cofactors are high-copy suppressors of

nab2-C437S                                                                                                          9

1.12 The RNA exosome subunits and cofactors are highly conserved              10

1.13 The RNA exosome subunits and cofactors are implicated in multiple

human diseases                                                                                                    11

1.14 Impairment of the RNA exosome suppresses the cold-sensitive growth

phenotype of nab2-C437S cells                                                                            12

1.15 Scope and potential implications of this project                                                    13


Chapter 2: The RNA binding protein Nab2 genetically interacts with multiple

RNA exosome cofactors to regulate target RNAs                                           17

2.1 Abstract                                                                                                           18

2.2 Introduction                                                                                                     20

2.3 Results                                                                                                            23

2.3.1 Specific RNA exosome cofactors suppress the growth defect of

nab2-C437S cells.                                                                                                 23

2.3.2 Suppression of the nab2-C437S cold-sensitive growth phenotype is

specific to the Nrd1 subunit of the NNS complex.                                                 24

2.3.3 The RNA binding function of Nrd1 is required for nab2-C437S suppression.  25

2.3.4 Suppression of the cold-sensitive growth phenotype is specific to Ski7 and

not subunits of the Ski complex.                                                                            26          

2.3.5 The interaction between Ski7 and the RNA exosome is required for

suppression of the nab2-C437S growth phenotype                                              27

2.3.6 Overexpression of suppressors results in altered transcript profiles.                   28

2.3.7 The nab2-C437S mutation affects a small subset of gene ontology

categories.                                                                                                            29

2.3.8 Overexpression of suppressors results in significant overlap of affected

transcripts and GO categories                                                                               30

2.3.9 Transcript levels altered by overexpression of suppressors are not altered

in wildtype control cells.                                                                                         31

2.3.10 Overexpression of NRD1 and SKI7 may suppress through partially

different mechanisms.                                                                                           32

2.4 Discussion                                                                                                       34


Chapter 3: The steady-state level of TLC1, the RNA scaffold component of S. cerevisiae telomerase, is decreased in nab2-C437S cells without affecting telomere length or lifespan.                                                                               58

3.1 Introduction                                                                                                     59

3.1.1 Telomeres protect chromosome ends from degradation.                        59

3.1.2 Telomerase and TLC1                                                                                  59

3.2 Results                                                                                                            61

3.2.1 Telomere maintenance genes have decreased steady-state levels in

nab2-C437S cells                                                                                                 61

3.2.2 Nab2 plays a role in TLC1 processing.                                                               61

3.2.3 Reduced TLC1 levels in nab2-C437S cells do not result in shorter telomeres

or cellular senescence.                                                                                         62

3.2.4 Deletion of RRP6 results in substantially increased levels of TLC1.            63

3.3 Discussion                                                                                                       65


Chapter 4: General Discussion                                                                          70

4.1 Recap and implications of this study                                                                                                                                                                                                                                                                                                                                                                                                                            71

4.2 Transcriptomic changes occurring from suppressor overexpression are broad

but subtle.                                                                                                                                                                                                                                                                                                                                                                                                                                                                          72

4.3 Nab2-C437S cells show RNA processing defects.                                                                                                                                                                                                                                                                                                                                      73

4.4 Overexpression of NRD1 results in RNA processing defects.                                                                                                                                                                            74                                                                    


4.5 AZF1 overexpression suppresses the nab2-C437S cold-sensitive growth

phenotype.                                                                                                                                                                                                                                                                                                                                                                                                                                                                         76

4.6 The environmental stress response does not explain altered steady-state

transcript levels by suppressors.                                                                                                                                                                                                                                                                                                                                                                                                                                       77

4.7 Potential mechanism of suppression by Mtr4                                                                                                                                                                                                                                                                                                                                           78

4.8 Potential mechanism of suppression by Nop8                                                                                                                                                                                                                                                                                                                                          80

4.9 A potential role for Rrp6 in suppression                                                                                                                                                                                                                                                                                                                                                                                                                       81

4.10 Concluding remarks and future directions                                                                                                                                                                                                                                                                                                                                                                                                       85


Chapter 5: Materials and Methods                                                                     93

5.1 Materials and Methods – Chapter 2                                                                94

5.2 Materials and Methods – Chapter 3                                                                98

Chapter 6: References                                                                                        100


List of Figures:

Figures - Chapter 1:                                                                                           15

Figure 1-1: ZC3H14 domains are evolutionarily conserved.                                  15

Figure 1-2: Structure of the S. cerevisiae nuclear RNA exosome                           16


Figures - Chapter 2:                                                                                            40

Figure 2-1: Suppression of the nab2-C437S cold-sensitive growth defect is

specific to RNA exosome cofactors SKI7, NRD1, NOP8, and MTR4.           40

Figure 2-2: Suppression is specific to NRD1 of the Nrd1-Nab3-Sen1 (NNS)

complex and requires the Nrd1 RNA binding domain.                                                  42

Figure 2-3: Suppression is specific to SKI7 and requires the Ski7-RNA exosome interacting domain.                                                                                              44

Figure 2-4: RNA-sequencing reveals distinct transcriptomes for nab2-C437S

cells overexpressed with suppressor genes.                                                              46

Figure 2-5: Nab2-C437S cells show 14 gene ontology categories significantly

down compared to control cells.                                                                            48

Figure 2-6: The top 14 down categories in nab2-C437S cells show disparate

patterns of rescue/lack of rescue upon overexpression of suppressors NRD1,

SKI7, and RRP41.                                                                                                 50

Figure 2-7: Overexpression of suppressors in nab2-C437S cells results in

significant overlap of differentially expressed genes between different

suppressor samples.                                                                                             51

Figure 2-8: Overexpression of NRD1, SKI7, and RRP41 results in significant

overlap of differentially expressed gene ontology categories affected by each suppressor.                                                                                                           52

Figure 2-9: Overexpression of suppressor genes in control cells affects

minimal genes compared to overexpression in nab2-C437S cells.                              54

Figure 2-10: Co-overexpression of NRD1 and SKI7 improves growth compared

to single overexpression but not more than RRP41 overexpression.                         55

Figure 2-11: Model: Overexpression of RNA exosome cofactors NRD1 and SKI7

impair nuclear RNA exosome function.                                                                 56                                                                                   

Figures - Chapter 3:                                                                                            67

Figure 3-1: TLC1 levels are decreased in nab2-C437S cells, and growth is

rescued by deletion of RRP6.                                                                                67

Figure 3-2: Nab2-C437S cells do not show shortened telomeres nor cellular

senescence.                                                                                                          68


Figures - Chapter 4:                                                                                            89

Figure 4-1: Overexpression of suppressors results in increased intronic reads in

a subset of transcripts.                                                                                          89

Figure 4-2: Overexpression of suppressors results in 5’ and/or 3’ end processing defects in a subset of transcripts.                                                                           90

Figure 4-3: Domain structure of Mtr4.                                                                   91

Figure 4-4: Domain structure of Nop8.                                                                  92

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