REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Rabbit polyclonal anti mCherry	Biovision	Cat#5993-100; RRID:AB_1975001
Mouse monoclonal anti PGK	Invitrogen	Cat#459250; RRID:AB_2532235
Goat IgG anti-Rabbit IgG (H+L)-Alexa Fluor 790	Jackson Immuno Research	Cat#111-655-144; RRID:AB_2338086
Goat anti-Mouse IgG (H+L)-Alexa Fluor 680	Dianova	Cat#115-625-146; RRID:AB_2338935
Bacterial and Virus Strains
Sf9 cells	Expression Systems	Cat#94-001F
Subcloning Efficiency DH5α Competent Cells	Invitrogen	Cat#18265017
NEB 5-alpha Competent E. coli	NEB	Cat#C2987I
E. coli XL1-Blue	Agilent	Cat#200249
Chemicals, Peptides, and Recombinant Proteins
RNaseOUT	Invitrogen	Cat#10777019
cOmplete Protease Inhibitor Cocktail	Roche	Cat#11836145001
cOmplete Protease Inhibitor Cocktail, EDTA-free	Roche	Cat#5056489001
Bond-Breaker TCEP Solution	ThermoFisher Scientific	Cat#77720
Magnesium acetate	Sigma	Cat#M-2545
Potassium acetate	Merck	Cat#104820
Potassium chloride	Merck	Cat#104935
HEPES	Carl Roth	Cat# 9105.2
DTT	Fermentas Life Sciences	Cat#R0862
ATP	Applichem	Cat#A1348,0005 5g
GTP	Thermo Fisher	Cat#18332015
Creatine phosphate	Merck	Cat#2380
Creatine phosphokinase	Sigma	Cat#C3755
Micrococcal nuclease	NEB	Cat#M0247S
2,4-dinitrophenol (DNP)	Sigma-Aldrich	Cat#D199303
Pipes	Applichem	Cat# A1079
Mannitol	Sigma	Cat#M4125
Zeba Desalt Spin columns	Thermo Fisher	Cat# 89894
Tris	Carl Roth	Cat# 5429
Triton X-100	Serva	Cat# 39795
Benzonase	In-house produced	N/A
Amylose resin	NEB	Cat# E8021S
EDTA	Roche	Cat# 105063
GST-3C Protease	(PreScission Protease; inhouse produced)	N/A
Acid-washed glass beads (425-600 μm)	Sigma-Aldrich	Cat#G8772
DNase I	Roche	Cat#4716728001
RNase I	Ambion	Cat#AM2295
25 μm polystyrene beads (Polybead)	Polysciences	Cat#30 μm polystyrene beads (Polybead)
Pico-SurfTM 1 (2% in NovecTM 7500)	Sphere Fluidics	Cat#F001
Sybr Green II	Invitrogen	Cat#S7564
T4 RNA ligase	NEB	Cat#M0204S
pCp-Cy3	Jena Bioscience	Cat#NU-1706-Cy3
MicroSpin G-25 columns	GE Healthcare Life Sciences	Cat#27532501
Ded1-monoGFP	This paper	N/A
Ded1ΔC-monoGFP	This paper	N/A
Ded1ΔN-monoGFP	This paper	N/A
Ded1_S.k.-monoGFP	This paper	N/A
Ded1_T.t.-monoGFP	This paper	N/A
Ded1-IDRm-monoGFP	This paper	N/A
Ded1	This paper	N/A
Pab1-monoGFP	This paper	N/A
monoGFP	In house produced	N/A
Critical Commercial Assays
mMESSAGE mMACHINE T7 Transcription Kit	ThermoFisher	Cat#AM1344
Poly(A) Tailing Kit	ThermoFisher	Cat#AM1350
Nano-Glo Luciferase Assay	Promega	Cat#N1110
QuikChange II Site-Directed Mutagenesis Kit	Agilent	Cat#200523
RNA 6000 Nano Kit	Agilent	Cat# 5067-1511
Q5 Site-Directed Mutagenesis Kit	NEB	Cat#E0554S
Deposited Data
5′UTR sequences	Kertesz et al., 2010	https://genie.weizmann.ac.il/pubs/PARS10/
Genome of S288C (SGD: R64-2-1)	Saccharomyces Genome Database	https://www.yeastgenome.org/
Genome of W303 GenBank: JRIU00000000)	Saccharomyces Genome Database	https://www.yeastgenome.org/
Ribosome profiling data (GEO: GSE131176)	This paper	https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE131176
RNA-Seq data (GEO: GSE141029)	This paper	https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE141029
Experimental Models: Organisms/Strains
W303 ADE+	Gift, Zachariae lab	#2952
W303 ded1::HIS::s.k.Ded1-sfGFP-KanMX	This paper	#4322
W303 ded1::HIS::ded1-sfGFP-KanMX	This paper	N/A
W303 ded1::HIS::T.t.Ded1-sfGFP-KanMX	This paper	#4255
W303 ADE+ Ded1-sfGFP-KanMX, Pab1-mCherry-Hyg	This paper	#4096
W303 ADE+ Ded1-mCherry-His3MX6	This paper	#4194
W303 ADE+ Ded1-sfGFP-KanMX	This paper	#4253
W303 ADE+ Ded1-IDR-mutant-mCherry-His3MX6	This paper	#4314
Oligonucleotides
Ribosomal RNA	Bioworld	Cat#11020001-2
tRNA	Invitrogen	Cat#AM7119
pUC57-kan-fw-seq: CCTCTTCGCTATTACGC	This paper	N/A
pUC57-kan-rev-seq: AGTTAGCTCACTCATTAGGC	This paper	N/A
Ded1-237-256-fw: ATCTAACGGCCGTTCTGGTG	This paper	N/A
Ded1-1663-1644-rev: CTCTGTTGTTGTTGCGACCG	This paper	N/A
Seq-Ded1-fw: CTAACGGTAGAGACTTGATGG	This paper	N/A
Ded1ΔN-NotI-fw-1: AGTACGCGGCCGCAACCGAA TTTACGTCTCCCCCTTTG	This paper	N/A
Ded1ΔN-rev: CTACGCATGCCTTAACCCATGATCTG	This paper	N/A
Ded1ΔC-fw: ACCCGACAGCTGTGATTATGGC	This paper	N/A
Ded1dC-AscI-rev: AGTCAGGCGCGCCCACTT CTTGGTTAGCCTCTGTCAGG	This paper	N/A
Ded1-C1-Guide-fw: CGGGTGGCGAATGGGACTTTTTTA CCTCACCTCCATTGGAGTTTTAGAGCTAGAAATAGC	This paper	N/A
Ded1-C1-Guide-rev: GCTATTTCTAGCTCTAAAACTCCA ATGGAGGTGAGGTAAAAAAGTCCCATTCGCCACCCG	This paper	N/A
Ded1-C1-mutant: GGACGTTTCTGGTAAGGATGTTCCT GAACCAATCACAGAATTTACCTCTCCTCCATTGGA	This paper	N/A
Ded1-C1-A-fw: AATTTCCAATCTTCTGGTATTAACTTCG ATAACTACGATGATATTCCAGTGGACGTTTCT	This paper	N/A
Ded1-C1-A-rev: GTGTTGGCTTGGTGAAACGGGCCAATTT GATGTTTTCCAATAACAATCCGTCCAATGGAG	This paper	N/A
Ded1-yeast-rv: ATCAGAGTTTGTCTTTCACC	This paper	N/A
Ded1-yeast-seq-rv: CAACAGGAGTCATATCACAG	This paper	N/A
Ded1-5UTR-amp-fw: CAGAGGCTAGCAGAATTACCCTCC	This paper	N/A
Ded1-5UTR-amp-rev: AATATGAAATGCTTT TCTTGTTGTTCTTACGG	This paper	N/A
Ded1-3UTR-amp-fw: GTGCTGGTGCTGGTTTAATTAACATG	This paper	N/A
Ded1-3UTR-amp-rev: TTTACACTTTATGCTTCCGGCTCGTATG	This paper	N/A
Ded1-T_t-amp-fw: TAGGTGGCAAGTGGTATTCCGTAAGAACA ACAAGAAAAGCATTTCATATTATGGCTGAGCAATTGTCAGGATC	This paper	N/A
Ded1-T_tamp-rev: ACAACACCAGTGAATAATTCTTCACCTTT AGACATGTTAATTAAACCAGCACCAGCACCC	This paper	N/A
Ded1-S_k-amp-fw: TAGGTGGCAAGTGGTATT CCGTAAGAACAACAAGAAAAGCATTTCATATT ATGGCTGAGCTGTCTGAACAGG	This paper	N/A
Ded1-sk-seq1-fw: CTGTGATAATGGCTCCTAC	This paper	N/A
Ded1-sk-seq2-fw: GCAGTCAGAGAGAGAAAG	This paper	N/A
Ded1-Sc-seq-c-term-fw: TGCATGAGATCTTGACTG	This paper	N/A
Ded1-seqc-fw: TGCATGAAATTTTGACTG	This paper	N/A
Ded1-5UTR-IVT-fw: ATTTCATATTATGGTTTT TACTTTAGAAGATTTTG	This paper	N/A
Ded1-5UTR-IVT-rev: GCTTTTCTTGTCCCTAT AGTGAGTCGTATTAG	This paper	N/A
Ded1-T_t-seq-fw: TGAGCAACATAGAACTAGC	This paper	N/A
Ded1-T_t-seq-fw2: TACGTCAGAGAATATCACC	This paper	N/A
Tt-Ded1-AR-seq3-fw: ATGGCTGAGCAATTGTCAG	This paper	N/A
Tt-Ded1-AR-seq4-rev: TTTCCACTACCAGTTTGAGC	This paper	N/A
Gis2-5UTR-IVT-fw: GAAAAAGATGGTTTTTA CTTTAGAAGATTTTG	This paper	N/A
Gis2-5UTR-IVT-rev: GTATTCTCCCTAT AGTGAGTCGTATTAG	This paper	N/A
Pab1-sense: GTCTTTCAAAAAGGAGCAAGAACAACAAA CTGAGCAAGCTGGTGACGGTGCTGGTTTA	This paper	N/A
Pab1-asense: TAAGTTTGTTGAGTAGGGAAGTAGGTGAT TACATAGAGCATCGATGAATTCGAGCTCG	This paper	N/A
tz_yor204w_35-53_guide_F: CGGGTGGCGAATGGGACTTT AACTACAATAACAACAACGGGTTTTAGAGCTAGAAATAGC	This paper	N/A
tz_yor204w_35-53_guide_R: GCTATTTCTAG CTCTAAAACCCGTTGTTGTTATTGTAGTTAAA GTCCCATTCGCCACCCG	This paper	N/A
tz_yor204w_35-53_sim_351_repair_mol: AGTGAGAACCTAAACAGGTTATTGATCT TCCTAAACCCTGAGTATTCAAGCAATCTTGGG	This paper	N/A
tz_yor204w_35-53_sim_351_repair_F: AGAAT GGTTATGTTCCTCCTCACTTAAGAGGAAAAC CAAGAAGTGCCAGAAGTGAGAACC	This paper	N/A
tz_yor204w_35-53_sim_351_repair_R: AAGAAAC CACCGTTGCCGTAACCACCACGACGGTTGTT GCTAAAGAAGCTCCCAAGATTG	This paper	N/A
Recombinant DNA
pKT127-sfGFP	This paper	L-269
pKT128-sfGFP	This paper	L-270
pBS35	Yeast Resource Center Washington	N/A
pKT128-mCherry	This paper	L-295
pUC57-kan-Pab1	Genscript	L-609
pUC57-kan-Ded1	Genscript	L-605
pUC57-kan-Ded1ΔC	This paper	L-606
pUC57-kan-T.t.-Ded1-sc_opt	Genscript	L-535
pUC57-kan-S.k.-Ded1-sc_opt	Genscript	L-536
pUC57-kan-Ded1-plus-for allele replacement	Genscript	L-534
pUC57-kan-T.t.-Ded1	Genscript	L-527
pUC57-kan-S.k.-Ded1	Genscript	L-528
pOCC120 Ded1ΔC	This paper	L-617
pOCC120-Pab1	This paper	L-612
pOCC97-Ded1	This paper	L-613
pOCC120-Ded1	This paper	L-604
pOCC177-kan-Ded1-S.k	This paper	L-558
pOCC120-kan-T.t.-Ded1	This paper	L-538
pOCC120-kan-S.k.-Ded1	This paper	L-539
pOCC120-Ded1ΔN	This paper	L-726
pOCC120-Ded1-IDRm	This paper	L-729
pUC57-kan-Ssa1-5UTR-Nanoluc	Genscript	L-638
pUC57-kan-Ssa4-5UTR-Nanoluc	Genscript	L-635
pUC57-kan-Hsp12-5UTR-Nanoluc	Genscript	L-652
pUC57-kan-Hsp104-5UTR-Nanoluc	Genscript	L-636
pUC57-kan-Ssk2-5UTR-Nanoluc	Genscript	L-639
pUC57-kan-Sbe22-5UTR-Nanoluc	Genscript	L-646
pUC57-kan-Pab1-5UTR-Nanoluc	Genscript	L-645
pUC57-kan-Gis2-5UTR-Nanoluc	This paper	L-647
pUC57-kan-Ded1-5UTR-Nanoluc	This paper	L-649
pCas	Ryan et al., 2016	Addgene plasmid # 60847
Software and Algorithms
Fiji	NIH	https://fiji.sc/
KNIME	KNIME.com AG	https://www.knime.com
FastQC (v0.11.2)	Babraham Institute	https://www.bioinformatics.babraham.ac.uk/projects/fastqc/
R / RStudio	R Core Team	https://www.r-project.org/ https://www.rstudio.com/
cutadapt (v1.17)	PyPI	https://cutadapt.readthedocs.io/en/stable/installation.html
ClustalW	EMBL-EBI	https://www.ebi.ac.uk/Tools/msa/clustalw2/
STAR 2.5.2b	GitHub	https://github.com/alexdobin/STAR/releases
PR. ThermControl software	Nanotemper	https://nanotempertech.com/prometheus-pr-thermcontrol-software/
DESeq2 (v1.22.2)	Bioconductor	https://bioconductor.org/packages/release/bioc/html/DESeq2.html
clusterProfiler (v3.10.1)	Bioconductor	http://bioconductor.org/packages/release/bioc/html/clusterProfiler.html
RNAfold (ViennaRNA Package v2.4.11)	TBI (University of Vienna)	https://www.tbi.univie.ac.at/RNA/
Bowtie 2 (v2.2.6)	bioconda	https://anaconda.org/bioconda/bowtie2/files