High impact information on NPL3

• To ascertain the mechanism of this process, we show that RNA polymerase II transcription is sufficient to recruit the Saccharomyces cerevisiae hnRNP protein Npl3 to a gene independent of RNA sequence [1].
• Combined mutations in the Saccharomyces cerevisiae hnRNP Npl3 and TATA-binding protein (TBP) block mRNA export, implying that cotranscriptional recruitment of Npl3 is required for efficient export of mRNA [2].
• This import defect can be reversed by increasing the intracellular concentration of Mtr10p, the nuclear import receptor for Npl3p [3].
• Interestingly, following induction of stress by heat shock, high salt, or ethanol, conditions under which most mRNA export is blocked, Npl3p is still exported from the nucleus [3].
• Hmt1p methylates both Npl3p and Hrp1p, which are shuttling hnRNPs involved in mRNA processing and export [4].

Biological context of NPL3

• HMT1 is not required for normal cell viability except when NPL3 is also defective [5].
• Changes in the methylation status of Npl3p do not correlate with its nucleocytoplasmic distribution [6].
• A crm1 mutant that inhibits export of proteins with leucine-rich nuclear export signals and mRNAs does not inhibit Npl3p export [6].
• The discovery of two hnRNP homologues that can partially suppress the function of Npl3p, also an RNA binding protein, will be discussed in terms of the possible roles for Npl3p in RNA metabolism [7].
• Our genetic analysis indicates that NPL3 is essential for normal cell growth; cells lacking NPL3 are temperature sensitive for growth but do not exhibit a defect in localization of nuclear proteins [8].

Anatomical context of NPL3

• Although this protein is not concentrated in nuclear pores, NPL3 is implicated in both import and export from the nucleus [9].
• Interestingly, a prolonged association of Npl3p with polysome containing mRNAs results in translational defects, indicating that Npl3p can function as a negative translational regulator [10].
• In cells depleted of NOP3, production of cytoplasmic ribosomes is impaired [11].
• These results suggest that nuclear import of Npl3p is coordinately influenced by methylation and phosphorylation in budding yeast, which may represent conserved components in the dynamic regulation of RNA processing in higher eukaryotic cells [12].

Associations of NPL3 with chemical compounds

• In contrast, arginine methylation is required for the export of the nuclear RNA-binding proteins Npl3p, Hrp1p, and Nab2p [13].
• A conditional lethal mutation, GAL::nop3, was constructed; growth of the mutant strain in glucose medium represses NOP3 expression [11].
• We constructed a set of Npl3 proteins in which subsets of the RGG arginines were mutated to lysine [14].
• The protein is similar to the other two serine/arginine (SR)-type proteins in yeast, Gbp2 and Npl3 [15].
• Amino acid changes in the putative Hmt1p S-adenosyl-L-methionine-binding site were generated and shown to be unable to catalyze methylation of Npl3p in vitro and in vivo or to restore growth to strains that require HMT1 [16].

Physical interactions of NPL3

• However, cells missing HMT1 and also bearing mutations in the mRNA-binding proteins Npl3p or Cbp80p can no longer survive, providing genetic backgrounds in which to study Hmt1p function [16].
• Purified Mtr10p forms a complex with Npl3p, an RNA-binding protein that shuttles in and out of the nucleus [17].

Enzymatic interactions of NPL3

• Sky1p bound and phosphorylated Npl3 with a Km that was 2 orders of magnitude lower than a short peptide mimic representing the phosphorylation site and only proximal determinants [18].

Regulatory relationships of NPL3

• Mutations in HRP1 suppress all npl3 temperature-sensitive alleles but do not bypass an npl3 null allele [7].
• Phosphorylation by Sky1p promotes Npl3p shuttling and mRNA dissociation [19].
• These mutations also resulted in a decreased, methylation-independent interaction of Npl3 with transcription elongation factor Tho2 and inhibited Npl3 self-association [14].
• We present evidence that the essential, nuclear phosphatase Glc7p promotes dephosphorylation of Npl3p in vivo and that nuclear dephosphorylation of Npl3p is required for mRNA export [20].

Other interactions of NPL3

• The HRP1 gene was originally isolated as a suppressor of a temperature-sensitive npl3 allele, a gene encoding a protein involved in mRNA export [21].
• Moreover, several proteins needed for Npl3p export are not needed for export of a typical Crm1p cargo [6].
• We demonstrate that Npl3p is methylated by Hmt1p both in vivo and in vitro [5].
• One of these is the previously known RNA15, which, like NPL3, also encodes a protein with similarity to the vertebrate hnRNP A/B protein family [7].
• Roles of ABF1, NPL3, and YCL54 in silencing in Saccharomyces cerevisiae [22].
• Npl3 competes with Rna15 for binding to a polyadenylation precursor and inhibits cleavage and polyadenylation in vitro [23].

Analytical, diagnostic and therapeutic context of NPL3

• Further evidence of an interaction between Npl3p and the cap-binding complex was revealed by co-immunoprecipitation experiments; Cbp80p and Cbp20p specifically co-precipitate with Npl3p [24].
• NOP3-specific antibodies recognize a 60-kD protein by SDS-PAGE and decorate the nucleolus and the surrounding nucleoplasm [11].
• Finally, Npl3 is recruited to genes in a transcription dependent manner as determined by chromatin immunoprecipitation [2].
• We have identified a temperature-sensitive mutant of Saccharomyces cerevisiae (npl3) that accumulates polyadenylated RNA in the nucleus at 37 degrees C, as judged by in situ hybridization [9].
• As judged by immunofluorescence and in situ hybridization, a temperature-sensitive allele of nop3 leads to the accumulation of poly(A)+ RNA in the nucleus at non-permissive temperature [25].

References