Nuclear Trafficking of La Protein Depends on a Newly Identified Nucleolar Localization Signal and the Ability to Bind RNA

Abstract

Here we provide evidence for an interaction-dependent subnuclear trafficking of the human La (hLa) protein, known as transient interaction partner of a variety of RNAs. Among these, precursor transcripts of certain RNAs are located in the nucleoplasm or nucleolus. Here we examined which functional domains of hLa are involved in its nuclear trafficking. By using green fluorescent-hLa fusion proteins, we discovered a nucleolar localization signal and demonstrated its functionality in a heterologous context. In addition, we revealed that the RRM2 motif of hLa is essential both for its RNA binding competence in vitro and in vivo and its exit from the nucleolus. Our data imply that hLa traffics between different subnuclear compartments, which depend decisively on a functional nucleolar localization signal as well as on RNA binding. Directed trafficking of hLa is fully consistent with its function in the maturation of precursor RNAs located in different subnuclear compartments.The human La protein (hLa) 1 is a 47-kDa phosphoprotein predominantly localized in the nucleus. It was first discovered as an autoantigen recognized by antibodies present in the sera of patients suffering from systemic lupus erythematosus and Sjogren's syndrome (1, 2). The La protein is a member of a large group of RNA-binding proteins containing RNA recognition motifs (RRMs) (3-7) and interacts with a variety of small RNAs (for reviews see Refs. 8 -10). The La protein is implicated in several aspects of the RNA metabolism, including processing of precursors of tRNAs, U3 snoRNA, U1 RNA, U6 snRNA (11)(12)(13)(14)(15)(16)(17)(18)(19)(20), and stabilization of viral (21-23) as well as cellular RNAs (24). In the cytoplasm, La has been implicated in the translational control of viral (25-27) and cellular RNAs (28 -30).The human La protein contains a nuclear localization sequence at the very C-terminal end, and the staining for endogenous La as well as the distribution of GFP-tagged La reveal a predominantly diffuse nuclear staining, although this may appear different under certain conditions or treatments (31-33). Subcellular distribution of human and yeast La was assumed to be independent of phosphorylation (34, 35); however, recent studies (36, 37) revealed that phosphorylation at Ser-366 may well influence subcellular localization of La. A regulated, nonhomogeneous nuclear distribution of La may also be predicted from the distinct nuclear distribution of its various RNA interaction partners. For instance, pre-tRNAs are most likely synthesized in the nucleoplasm (38) but are processed, at least in part, in the nucleolus (39, 40), whereas in yeast the La-associated precursor of U3 snoRNA is probably processed in the nucleolar body and nucleolus (20). Nevertheless, interactions with precursor RNAs in different nuclear compartments implicate a highly dynamic and regulated nuclear distribution of La. Although a nucleolar localization of La has been reported (for review see Ref. 9), the hLa protein was not identified among the recently resolved protein ...

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