Histone methyltransferase SUV39H1 regulates the Golgi complex via the nuclear envelope-spanning LINC complex.

Cell motility is related to the higher-order structure of chromatin. Stimuli that induce cell migration change chromatin organization; such stimuli include elevated histone H3 lysine 9 trimethylation (H3K9me3). We previously showed that depletion of histone H3 lysine 9 methyltransferase, SUV39H1, suppresses directional cell migration. However, the molecular mechanism underlying this association between chromatin and cell migration remains elusive. The Golgi apparatus is a cell organelle essential for cell motility. In this study, we show that loss of H3K9 methyltransferase SUV39H1 but not SETDB1 or SETDB2 causes dispersion of the Golgi apparatus throughout the cytoplasm. The Golgi dispersion triggered by SUV39H1 depletion is independent of transcription, centrosomes, and microtubule organization, but is suppressed by depletion of any of the following three proteins: LINC complex components SUN2, nesprin-2, or microtubule plus-end-directed kinesin-like protein KIF20A. In addition, SUN2 is closely localized to H3K9me3, and SUV39H1 affects the mobility of SUN2 in the nuclear envelope. Further, inhibition of cell motility caused by SUV39H1 depletion is restored by suppression of SUN2, nesprin-2, or KIF20A. In summary, these results show the functional association between chromatin organization and cell motility via the Golgi organization regulated by the LINC complex.

Nsp14 of SARS-CoV-2 inhibits mRNA processing and nuclear export by targeting the nuclear cap-binding complex.

To facilitate selfish replication, viruses halt host gene expression in various ways. The nuclear export of mRNA is one such process targeted by many viruses. SARS-CoV-2, the etiological agent of severe acute respiratory syndrome, also prevents mRNA nuclear export. In this study, Nsp14, a bifunctional viral replicase subunit, was identified as a novel inhibitor of mRNA nuclear export. Nsp14 induces poly(A)+ RNA nuclear accumulation and the dissolution/coalescence of nuclear speckles. Genome-wide gene expression analysis revealed the global dysregulation of splicing and 3'-end processing defects of replication-dependent histone mRNAs by Nsp14. These abnormalities were also observed in SARS-CoV-2-infected cells. A mutation introduced at the guanine-N7-methyltransferase active site of Nsp14 diminished these inhibitory activities. Targeted capillary electrophoresis-mass spectrometry analysis (CE-MS) unveiled the production of N7-methyl-GTP in Nsp14-expressing cells. Association of the nuclear cap-binding complex (NCBC) with the mRNA cap and subsequent recruitment of U1 snRNP and the stem-loop binding protein (SLBP) were impaired by Nsp14. These data suggest that the defects in mRNA processing and export arise from the compromise of NCBC function by N7-methyl-GTP, thus exemplifying a novel viral strategy to block host gene expression.

Inner Nuclear Membrane Protein, SUN1, is Required for Cytoskeletal Force Generation and Focal Adhesion Maturation.

The linker of nucleoskeleton and cytoskeleton (LINC) complex is composed of the inner nuclear membrane-spanning SUN proteins and the outer nuclear membrane-spanning nesprin proteins. The LINC complex physically connects the nucleus and plasma membrane via the actin cytoskeleton to perform diverse functions including mechanotransduction from the extracellular environment to the nucleus. Mammalian somatic cells express two principal SUN proteins, namely SUN1 and SUN2. We have previously reported that SUN1, but not SUN2, is essential for directional cell migration; however, the underlying mechanism remains elusive. Because the balance between adhesive force and traction force is critical for cell migration, in the present study, we focused on focal adhesions (FAs) and the actin cytoskeleton. We observed that siRNA-mediated SUN1 depletion did not affect the recruitment of integrin ß1, one of the ubiquitously expressed focal adhesion molecules, to the plasma membrane. Consistently, SUN1-depleted cells normally adhered to extracellular matrix proteins, including collagen, fibronectin, laminin, and vitronectin. In contrast, SUN1 depletion reduced the activation of integrin ß1. Strikingly, the depletion of SUN1 interfered with the incorporation of vinculin into the focal adhesions, whereas no significant differences in the expression of vinculin were observed between wild-type and SUN1-depleted cells. In addition, SUN1 depletion suppressed the recruitment of zyxin to nascent focal adhesions. These data indicate that SUN1 is involved in the maturation of focal adhesions. Moreover, disruption of the SUN1-containing LINC complex abrogates the actin cytoskeleton and generation of intracellular traction force, despite the presence of SUN2. Thus, a physical link between the nucleus and cytoskeleton through SUN1 is required for the proper organization of actin, thereby suppressing the incorporation of vinculin and zyxin into focal adhesions and the activation of integrin ß1, both of which are dependent on traction force. This study provides insights into a previously unappreciated signaling pathway from the nucleus to the cytoskeleton, which is in the opposite direction to the well-known mechanotransduction pathways from the extracellular matrix to the nucleus.

Liquid chromatography-tandem mass spectrometry analysis of oxidation of 2'-, 3'-, 4'- and 6-hydroxyflavanones by human cytochrome P450 enzymes.

2'-Hydroxyflavanone (2'OHFva), 3'OHFva, 4'OHFva, and 6OHFva, the major oxidative products of flavanone by human cytochrome P450 (P450, CYP) enzymes, were studied in regard to further oxidation by human CYP1A1, 1A2, 1B1.1, 1B1.3, and 2A6. The products formed were analyzed with LC-MS/MS and characterized by their positive ion fragmentations on mass spectrometry. Several di-hydroxylated flavanone (diOHFva) and di-hydroxylated flavone (diOHFvo) products, detected by analyzing parent ions at m/z 257 and 255, respectively, were found following incubation of these four hydroxylated flavanones with P450s. The m/z 257 products were produced at higher levels than the latter with four substrates examined. The structures of the m/z 257 products were characterized by LC-MS/MS product ion spectra, and the results suggest that 3'OHFva and 4'OHFva are further oxidized mainly at B-ring by P450s while 6OHFva oxidation was at A-ring. Different diOHFvo products (m/z 255) were also characterized by LC-MS/MS, and the results suggested that most of these diOHFvo products were formed through oxidation or desaturation of the diOHFva products (m/z 257) by P450s. Only when 4'OHFva (m/z 241) was used as a substrate, formation of 4'OHFvo (m/z 239) was detected, indicating that diOHFvo might also be formed through oxidation of 4'OHFvo by P450s. Finally, our results indicated that CYP1 family enzymes were more active than CYP2A6 in catalyzing the oxidation of these four hydroxylated flavanones, and these findings were supported by molecular docking studies of these chemicals with active sites of P450 enzymes.

The SUN1 splicing variants SUN1_888 and SUN1_916 differentially regulate nucleolar structure.

The nucleolar structure is highly dynamic and strictly regulated in response to internal cues, such as metabolic rates, and to external cues, such as mechanical forces applied to cells. Although the multilayered nucleolar structure is largely determined by the liquid-like properties of RNA and proteins, the mechanisms regulating the morphology and number of nucleoli remain elusive. The linker of the nucleoskeleton and cytoskeleton (LINC) complex comprises inner nuclear membrane Sad1/UNC-84 (SUN) proteins and outer nuclear membrane-localized nesprins. We previously showed that the depletion of SUN1 proteins affects nucleolar morphologies. This study focuses on the function of SUN1 splicing variants in determining nucleolar morphology. An RNA interference strategy showed that the predominantly expressed variants, SUN1_888 and SUN1_916, were crucial for nucleolar morphology but functionally distinct. In addition, the depletion of either SUN1_888 or SUN1_916 altered the chromatin structure and affected the distribution of histone modifications. Based on these results, we propose a model in which the LINC complex plays a role in modulating nucleolar morphology and numbers via chromatin.

Preference for O-demethylation reactions in the oxidation of 2'-, 3'-, and 4'-methoxyflavones by human cytochrome P450 enzymes.

2'-, 3'-, and 4'-Methoxyflavones (MeFs) were incubated with nine forms of recombinant human cytochrome P450 (P450 or CYP) enzymes in the presence of an NADPH-generating system and the products formed were analyzed with LC-MS/MS methods.CYP1B1.1 and 1B1.3 were highly active in demethylating 4'MeF to form 4'-hydroxyflavone (rate of 5.0 nmol/min/nmol P450) and further to 3',4'-dihydroxyflavone (rates of 2.1 and 0.66 nmol/min/nmol P450, respectively). 3'MeF was found to be oxidized by P450s to m/z 239 (M-14) products (presumably 3'-hydroxyflavone) and then to 3',4'-dihydroxyflavone. P450s also catalyzed oxidation of 2'MeF to m/z 239 (M-14) and m/z 255 (M-14, M-14 + 16) products, presumably mono- and di-hydroxylated products, respectively.At least two types of ring oxidation products having m/z 269 fragments were formed, although at slower rates than the formation of mono- and di-hydroxylated products, on incubation of these MeFs with P450s; one type was products oxidized at the C-ring, having m/z 121 fragments, and the other one was the products oxidized at the A-ring (having m/z 137 fragments).Molecular docking analysis indicated the preference of interaction of O-methoxy moiety of methoxyflavones in the active site of CYP1A2.These results suggest that 2'-, 3'-, and 4'-methoxyflavones are principally demethylated by human P450s to form mono- and di-hydroxyflavones and that direct oxidation occurs in these MeFs to form mono-hydroxylated products, oxidized at the A- or B-ring of MeF.

Human THO maintains the stability of repetitive DNA.

The evolutionarily conserved multiprotein complex THO/TREX is required for pre-mRNA processing, mRNA export and the maintenance of genome stability. In this study, we analyzed the genome-wide distribution of human THOC7, a component of human THO, by chromatin immunoprecipitation sequencing. The analysis revealed that human THOC7 occupies repetitive sequences, which include microsatellite repeats in genic and intergenic regions and telomeric repeats. The majority of the THOC7 ChIP peaks overlapped with those of the elongating form of RNA polymerase II and R-loops, indicating that THOC7 accumulates in transcriptionally active repeat regions. Knocking down THOC5, an RNA-binding component of human THO, by siRNA induced the accumulation of γH2AX in the repeat regions. We also observed an aberration in the telomeres in the THOC5-depleted condition. These results suggest that human THO restrains the transcription-associated instability of repeat regions in the human genome.

Oxidation of Flavone, 5-Hydroxyflavone, and 5,7-Dihydroxyflavone to Mono-, Di-, and Tri-Hydroxyflavones by Human Cytochrome P450 Enzymes.

Biologically active plant flavonoids, including 5,7-dihydroxyflavone (57diOHF, chrysin), 4',5,7-trihydroxyflavone (4'57triOHF, apigenin), and 5,6,7-trihydroxyflavone (567triOHF, baicalein), have important pharmacological and toxicological significance, e.g., antiallergic, anti-inflammatory, antioxidative, antimicrobial, and antitumorgenic properties. In order to better understand the metabolism of these flavonoids in humans, we examined the oxidation of flavone, 5-hydroxyflavone (5OHF), and 57diOHF to various products by human cytochrome P450 (P450 or CYP) and liver microsomal enzymes. Individual human P450s and liver microsomes oxidized flavone to 6-hydroxyflavone, small amounts of 5OHF, and 11 other monohydroxylated products at different rates and also produced several dihydroxylated products (including 57diOHF and 7,8-dihydroxyflavone) from flavone. We also found that 5OHF was oxidized by several P450 enzymes and human liver microsomes to 57diOHF and further to 567triOHF, but the turnover rates in these reactions were low. Interestingly, both CYP1B1.1 and 1B1.3 converted 57diOHF to 567triOHF at turnover rates (on the basis of P450 contents) of >3.0 min-1, and CYP1A1 and 1A2 produced 567triOHF at rates of 0.51 and 0.72 min-1, respectively. CYP2A13 and 2A6 catalyzed the oxidation of 57diOHF to 4'57triOHF at rates of 0.7 and 0.1 min-1, respectively. Our present results show that different P450s have individual roles in oxidizing these phytochemical flavonoids and that these reactions may cause changes in their biological and toxicological properties in mammals.

Human THO coordinates transcription termination and subsequent transcript release from the HSP70 locus.

A multiprotein complex, THO/TREX, couples the transcription, 3'-end formation and nuclear export of mRNAs. In this study, we report that crucial factors for mRNA processing, such as XRN2, DDX5/DDX17 and CstF64, are copurified with human THO (hTHO). Using chromatin immunoprecipitation, we found increased cross-linking of XRN2 and CstF64 to the RNA polymerase II (RNAP II) pause site of the HSPA1A gene upon down-regulation of THOC5, a metazoan-specific component of hTHO. As observed in THOC5-depleted cells, knockdown of XRN2 blocked HSP70 transcript release and increased the amount of CstF64 at the pause site. In addition, our data indicate that DDX5/DDX17 is also required for HSP70 transcript release. As the degradation of read-through transcripts, but not cleavage at polyadenylation sites per se, was hindered upon THOC5 or DDX5/DDX17 down-regulation, these factors appear to influence transcriptional termination. Interestingly, over-expression of RNase H suppressed the accumulation of HSP70 transcripts in nuclear foci in THOC5- or DDX5/DDX17-depleted cells. Thus, we propose a model in which hTHO, along with DDX5/DDX17, restricts the formation of R-loops, thereby facilitating the XRN2-mediated transcriptional termination and release of the mature transcript from the HSPA1A locus.

Site-specific oxidation of flavanone and flavone by cytochrome P450 2A6 in human liver microsomes.

The roles of human cytochrome P450 (P450 or CYP) 2A6 in the oxidation of flavanone [(2R)- and (2S)-enantiomers] and flavone were studied in human liver microsomes and recombinant human P450 enzymes. CYP2A6 was highly active in oxidizing flavanone to form flavone, 2'-hydroxy-, 4'-, and 6-hydroxyflavanones and in oxidizing flavone to form mono- and di-hydroxylated products, such as mono-hydroxy flavones M6, M7, and M11 and di-hydroxy flavones M3, M4, and M5. Liver microsomes prepared from human sample HH2, defective in coumarin 7-hydroxylation activity, were very inefficient in forming 2'-hydroxyflavanone from flavanone and a mono-hydroxylated product, M6, from flavone. Coumarin and anti-CYP2A6 antibodies strongly inhibited the formation of these metabolites in microsomes prepared from liver samples HH47 and 54, which were active in coumarin oxidation activities. Molecular docking analysis showed that the C2'-position of (2R)-flavanone (3.8 Å) was closer to the iron center of CYP2A6 than the C6-position (10 Å), while distances from C2' and C6 of (2S)-flavanone to the CYP2A6 were 6.91 Å and 5.42 Å, respectively. These results suggest that CYP2A6 catalyzes site-specific oxidation of (racemic) flavanone and also flavone in human liver microsomes. CYP1A2 and CYP2B6 were also found to play significant roles in some of the oxidations of these flavonoids by human liver microsomes.

Relationship between PIWIL4-Mediated H3K4me2 Demethylation and piRNA-Dependent DNA Methylation.

Retrotransposon genes are silenced by DNA methylation because of potential harm due to insertional mutagenesis. DNA methylation of retrotransposon genes is erased and re-established during male germ cell development. Both piRNA-dependent and piRNA-independent mechanisms are active during the re-establishment process, with the piRNA-independent mechanism occurring first. In this study, we analyzed the role of PIWIL4/MIWI2 in the modification of histone H3 and subsequent piRNA-dependent DNA methylation. Dimethylation at H3K4 is highly enriched at piRNA-dependent methylated regions and anti-correlated with de novo DNA methylation during the phase of piRNA-independent DNA methylation. In addition, PIWIL4, which binds the H3K4 demethylases KDM1A and KDM5B, is required for removing H3K4me2 marks. These data show that PIWIL4 plays important roles in histone modification and piRNA-dependent DNA methylation.

SUN1 splice variants, SUN1_888, SUN1_785, and predominant SUN1_916, variably function in directional cell migration.

The LINC complex is a multifunctional protein complex that is involved in various processes at the nuclear envelope, such as nuclear migration, mechanotransduction and chromatin tethering in the meiotic phase. However, it remains unknown how these functions are regulated in different cell contexts. An inner nuclear membrane component of the LINC complex, SUN1, is ubiquitously expressed. The human SUN1 gene produces over 10 variants by alternative splicing. Although functions of SUN1 are relatively well characterized, functional differences among SUN1 splice variants are poorly characterized. LINC complex components are associated with a wide range of human diseases; therefore, it is important to understand the functional diversity among SUN1 splice variants. Here, we identified a novel human SUN1 splice variant, SUN1_888. overexpression of the SUN1 splice variants, SUN1_888 or SUN1_785, but not the predominant isoform, SUN1_916, activated directional cell migration. Knockdown of SUN1_888 suppressed cell migration; in contrast depletion of SUN1_916 activated cell migration. In addition, all of investigated SUN1 splicing variants rescued cell migration in SUN1 knock out cell. These results indicate that redundant and non-redundant functions of SUN1 splice variant in directional cell migration and suggest that variable LINC complexes with distinct task may exit. Furthermore, in contrast to previous studies, we showed association between SUN1 and B-type lamins. Interestingly, B-type lamin preferentially interacts with SUN1 but not SUN2. These results suggest that tissue-specific SUN1 variants variably interact with nucleoplasmic partners and allow variable assembly of LINC complexes that can be assigned to distinct tasks.

Loss of the integral nuclear envelope protein SUN1 induces alteration of nucleoli.

A supervised machine learning algorithm, which is qualified for image classification and analyzing similarities, is based on multiple discriminative morphological features that are automatically assembled during the learning processes. The algorithm is suitable for population-based analysis of images of biological materials that are generally complex and heterogeneous. Here we used the algorithm wndchrm to quantify the effects on nucleolar morphology of the loss of the components of nuclear envelope in a human mammary epithelial cell line. The linker of nucleoskeleton and cytoskeleton (LINC) complex, an assembly of nuclear envelope proteins comprising mainly members of the SUN and nesprin families, connects the nuclear lamina and cytoskeletal filaments. The components of the LINC complex are markedly deficient in breast cancer tissues. We found that a reduction in the levels of SUN1, SUN2, and lamin A/C led to significant changes in morphologies that were computationally classified using wndchrm with approximately 100% accuracy. In particular, depletion of SUN1 caused nucleolar hypertrophy and reduced rRNA synthesis. Further, wndchrm revealed a consistent negative correlation between SUN1 expression and the size of nucleoli in human breast cancer tissues. Our unbiased morphological quantitation strategies using wndchrm revealed an unexpected link between the components of the LINC complex and the morphologies of nucleoli that serves as an indicator of the malignant phenotype of breast cancer cells.

Nuclear export of messenger RNA.

Transport of messenger RNA (mRNA) from the nucleus to the cytoplasm is an essential step of eukaryotic gene expression. In the cell nucleus, a precursor mRNA undergoes a series of processing steps, including capping at the 5' ends, splicing and cleavage/polyadenylation at the 3' ends. During this process, the mRNA associates with a wide variety of proteins, forming a messenger ribonucleoprotein (mRNP) particle. Association with factors involved in nuclear export also occurs during transcription and processing, and thus nuclear export is fully integrated into mRNA maturation. The coupling between mRNA maturation and nuclear export is an important mechanism for providing only fully functional and competent mRNA to the cytoplasmic translational machinery, thereby ensuring accuracy and swiftness of gene expression. This review describes the molecular mechanism of nuclear mRNA export mediated by the principal transport factors, including Tap-p15 and the TREX complex.

NTF2-like domain of Tap plays a critical role in cargo mRNA recognition and export.

Metazoan Tap-p15 (also called Nxf1-Nxt1) and yeast Mex67-Mtr2 heterodimers are the general mRNA export receptors. The RNA binding activity of Tap-p15, which is essential for mRNA nuclear export, has been attributed to the amino-terminal RNA binding module of Tap consists of RNA recognition motif (RRM) and leucine-rich repeat. In this study, we identified a novel RNA interaction surface in the NTF2-like (NTF2L) domain of Tap, which is analogous to the rRNA binding platform of Mex67-Mtr2. Tap-p15 uses the three domains to tightly bind the retroviral constitutive transport element. The RNA binding through the NTF2L domain is functionally relevant as introduction of mutations in this region reduced CTE-containing mRNA export activity. In contrast, only when the RRM and NTF2L domains were mutated simultaneously, bulk poly (A)(+) RNA export and in vivo poly (A)(+) RNA binding activities of Tap-p15 were significantly attenuated. Moreover, an engineered human cell line harboring the NTF2L domain mutation in the NXF1 gene showed a synthetic growth phenotype and severe mRNA export defect under Aly/REF and Thoc5 depleted condition. These data suggest that Tap-p15 recognizes bulk mRNAs through combinatorial use of the distinct RNA binding domains.

The principal mRNA nuclear export factor NXF1:NXT1 forms a symmetric binding platform that facilitates export of retroviral CTE-RNA.

The NXF1:NXT1 complex (also known as TAP:p15) is a general mRNA nuclear export factor that is conserved from yeast to humans. NXF1 is a modular protein constructed from four domains (RRM, LRR, NTF2-like and UBA domains). It is currently unclear how NXF1:NXT1 binds transcripts and whether there is higher organization of the NXF1 domains. We report here the 3.4 Å resolution crystal structure of the first three domains of human NXF1 together with NXT1 that has two copies of the complex in the asymmetric unit arranged to form an intimate domain-swapped dimer. In this dimer, the linkers between the NXF1 LRR and NTF2-like domains interact with NXT1, generating a 2-fold symmetric platform in which the RNA-binding RRM, LRR and NTF2-like domains are arranged on one face. In addition to bulk transcripts, NXF1:NXT1 also facilitates the export of unspliced retroviral genomic RNA from simple type-D retroviruses such as SRV-1 that contain a constitutive transport element (CTE), a cis-acting 2-fold symmetric RNA stem-loop motif. Complementary structural, biochemical and cellular techniques indicated that the formation of a symmetric RNA binding platform generated by dimerization of NXF1:NXT1 facilitates the recognition of CTE-RNA and promotes its nuclear export.

Human TREX component Thoc5 affects alternative polyadenylation site choice by recruiting mammalian cleavage factor I.

The transcription-export complex (TREX) couples mRNA transcription, processing and nuclear export. We found that CFIm68, a large subunit of a heterotetrameric protein complex mammalian cleavage factor I (CFIm), which is implicated in alternative polyadenylation site choice, co-purified with Thoc5, a component of human TREX. Immunoprecipitation using antibodies against different components of TREX indicated that most likely both complexes interact via an interaction between Thoc5 and CFIm68. Microarray analysis using human HeLa cells revealed that a subset of genes was differentially expressed on Thoc5 knockdown. Notably, the depletion of Thoc5 selectively attenuated the expression of mRNAs polyadenylated at distal, but not proximal, polyadenylation sites, which phenocopied the depletion of CFIm68. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) indicated that CFIm68 preferentially associated with the 5' regions of genes; strikingly, the 5' peak of CFIm68 was significantly and globally reduced on Thoc5 knockdown. We suggest a model in which human Thoc5 controls polyadenylation site choice through the co-transcriptional loading of CFIm68 onto target genes.

The lipid mediator protectin D1 inhibits influenza virus replication and improves severe influenza.

Influenza A viruses are a major cause of mortality. Given the potential for future lethal pandemics, effective drugs are needed for the treatment of severe influenza such as that caused by H5N1 viruses. Using mediator lipidomics and bioactive lipid screen, we report that the omega-3 polyunsaturated fatty acid (PUFA)-derived lipid mediator protectin D1 (PD1) markedly attenuated influenza virus replication via RNA export machinery. Production of PD1 was suppressed during severe influenza and PD1 levels inversely correlated with the pathogenicity of H5N1 viruses. Suppression of PD1 was genetically mapped to 12/15-lipoxygenase activity. Importantly, PD1 treatment improved the survival and pathology of severe influenza in mice, even under conditions where known antiviral drugs fail to protect from death. These results identify the endogenous lipid mediator PD1 as an innate suppressor of influenza virus replication that protects against lethal influenza virus infection.

Synchronizing nuclear import of ribosomal proteins with ribosome assembly.

Ribosomal proteins are synthesized in the cytoplasm, before nuclear import and assembly with ribosomal RNA (rRNA). Little is known about coordination of nucleocytoplasmic transport with ribosome assembly. Here, we identify a transport adaptor, symportin 1 (Syo1), that facilitates synchronized coimport of the two 5S-rRNA binding proteins Rpl5 and Rpl11. In vitro studies revealed that Syo1 concomitantly binds Rpl5-Rpl11 and furthermore recruits the import receptor Kap104. The Syo1-Rpl5-Rpl11 import complex is released from Kap104 by RanGTP and can be directly transferred onto the 5S rRNA. Syo1 can shuttle back to the cytoplasm by interaction with phenylalanine-glycine nucleoporins. X-ray crystallography uncovered how the α-solenoid symportin accommodates the Rpl5 amino terminus, normally bound to 5S rRNA, in an extended groove. Symportin-mediated coimport of Rpl5-Rpl11 could ensure coordinated and stoichiometric incorporation of these proteins into pre-60S ribosomes.