APUM23, a nucleolar Puf domain protein, is involved in pre-ribosomal RNA processing and normal growth patterning in Arabidopsis

Abstract

SUMMARYPumilio, an RNA-binding protein that contains tandemly repeated Puf domains, is known to repress translational activity in early embryogenesis and polarized cells of non-plant species. Although Pumilio proteins have been characterized in many eukaryotes, their role in plants is unknown. In the present study, we characterized an Arabidopsis Pumilio-encoding gene, APUM23. APUM23 is constitutively expressed, with higher levels in metabolically active tissues, and its expression is up-regulated in the presence of either glucose or sucrose. The T-DNA insertion mutants apum23-1 and apum23-2 showed slow growth, with serrated and scrunched leaves, an abnormal venation pattern, and distorted organization of the palisade parenchyma cellsa phenotype that is reminiscent of nucleolin and ribosomal protein gene mutants. Intracellular localization studies indicate that APUM23 predominantly localizes to the nucleolus. Based on this localization, rRNA processing was examined. In apum23, 35S pre-rRNA, and unprocessed 18S and 5.8S poly(A) rRNAs, accumulated without affecting the steady-state levels of mature rRNAs, indicating that APUM23 is involved in the processing and/or degradation of 35S pre-rRNA and rRNA maturation by-products. The apum23 mutant showed increased levels of 18S rRNA biogenesis-related U3 and U14 small nucleolar RNAs (snoRNAs) and accumulated RNAs within the nucleolus. Our data suggest that APUM23 plays an important role in plant development via rRNA processing.

Arabidopsis protein arginine methyltransferase 3 is required for ribosome biogenesis by affecting precursor ribosomal RNA processing

“…These leaf developmental defects are reminiscent of the Arabidopsis mutants of ribosome proteins (40) (Table S1), like S18A (also known as POINTED FIRST LEAF, pfl in its mutant) in the small subunit (41), or L4A and L4D in the large subunit (42). Moreover, null mutants of many nucleolus-localized ribosome assembly factors (Table S1), including the Pumilio-like RNA binding protein ARABIDOPSIS PUMILIO 23 (APUM23) (24), nucleolar RNA helicase ARABIDOPSIS MRNA TRANSPORT 4 (AtMTR4) (23), and a putative methyltransferase ROOT INITI-ATION DEFECTIVE 2 (RID2) (43), also display similar developmental defects and aberrant pre-rRNA processing. In addition to the leaf phenotypes, atprmt3 mutants also displayed growth retardation, including delayed germination and primary root growth (Fig.…”

“…Then we focused on atprmt3-2 and AtPRMT3-GUS for further Northern blot assays. We used apum23-1 as a control mutant, because it shows aberrant accumulation of 35S and polyadenylated P-A3 pre-rRNAs (24). Probes used previously (22,23) were able to detect the 35S and 33S precursors (Fig.…”

“…The ribosome biogenesis mutants apum23 and rpl4 displayed varied sensitivity to translational inhibitors, as a result of putative aberrant ribosome structure and function (24,42). Therefore, we evaluated the ribosomal function in atprmt3 using antibiotics with known targets in the ribosome (44,45).…”

“…The cleavage at A3 in ITS1 occurs before complete removal of the 5′ ETS, generating diagnostic intermediates of P-A3 (22)(23)(24)(25) and P'-A3 fragments (23,25) in wild type. However, it remains unknown whether Arabidopsis has an alternative pre-rRNA processing pathway marked by 32S rRNA, resembling the "U3 dependent cleavage occurs first" pathway in budding yeast (17,26).…”

Pumilio Puf domain RNA-binding proteins in Arabidopsis

“…Any mutation in the ribosomal subunit genes affects auxin biosynthesis due to defective ribosomes, which translationally repress the auxin responsive factors, such cellular localization demonstrated that cytoplasmic punctuate structures are formed for many of the Arabidopsis Puf proteins, such as APUM7 (At1g78160), APUM8 (At1g22240), APUM9, APUM10, APUM12 (At5g56510), APUM14 (At5g43110) and APUM18. 44 However, others, i.e., APUM23 (At1g72320) and APUM24 (At3g16810), localize to the nucleolus, 44,45 suggesting that certain Puf proteins are involved in organelle-specific functions.…”