Domain architectures and characterization of an RNA-binding protein, TLS.
Translocated in liposarcoma (TLS) is an important protein component of the heterogeneous nuclear ribonucleoprotein complex involved in the splicing of pre-mRNA and the export of fully processed mRNA to the cytoplasm. We examined the domain organization of human TLS by a combined approach using limited proteolysis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, circular dichroism, inductively coupled plasma atomic emission spectroscopy, and NMR spectroscopy. We found that the RNA recognition motif (RRM) and zinc finger-like domains exclusively form protease-resistant core structures within the isolated TLS protein fragments, while the remaining regions, including the Arg-Gly-Gly repeats, appear to be completely unstructured. Thus, TLS contains the unstructured N-terminal half followed by the RRM and zinc finger-like domains, which are connected to each other by a flexible linker. We also carried out NMR analyses to obtain more detailed insights into the individual RRM and zinc finger-like domains. The 113Cd NMR analysis of the zinc finger-like domain verified that zinc is coordinated with four cysteines in the C4 type scheme. We also investigated the interaction of each domain with an oligo-RNA containing the GGUG sequence, which appears to be critical for the TLS function in splicing. The backbone amide NMR chemical shift perturbation analyses indicated that the zinc finger domain binds GGUG-containing RNA with a dissociation constant of about 1.0 x 10(-5) m, whereas the RRM domain showed no observable interaction with this RNA. This surprising result implies that the zinc finger domain plays a more predominant role in RNA recognition than the RRM domain.
Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan.
Yuko
Y
Iko
Takashi S
TS
Kodama
Nobuyuki
N
Kasai
Takuji
T
Oyama
Eugene H
EH
Morita
Takanori
T
Muto
Mika
M
Okumura
Ritsuko
R
Fujii
Toru
T
Takumi
Shin-ichi
S
Tate
Kosuke
K
Morikawa
D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000378:metabolism; D002918:Chymotrypsin / Q000494:pharmacology; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D012313:RNA / Q000737:chemistry; D034702:RNA-Binding Protein FUS / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013054:Spectrophotometry, Atomic; D016335:Zinc Fingers
D018076:DNA, Complementary; D011506:Proteins; D034702:RNA-Binding Protein FUS; D012313:RNA; D005982:Glutathione Transferase; D002918:Chymotrypsin; D003545:Cysteine