hLARP7 C-terminal domain contains an xRRM that binds the 3′ hairpin of 7SK RNA

Abstract

The 7SK small nuclear ribonucleoprotein (snRNP) sequesters and inactivates the positive transcription elongation factor b (P-TEFb), an essential eukaryotic mRNA transcription factor. The human La-related protein group 7 (hLARP7) is a constitutive component of the 7SK snRNP and localizes to the 3′ terminus of the 7SK long noncoding RNA. hLARP7, and in particular its C-terminal domain (CTD), is essential for 7SK RNA stability and assembly with P-TEFb. The hLARP7 N-terminal La module binds and protects the 3′ end from degradation, but the structural and functional role of its CTD is unclear. We report the solution NMR structure of the hLARP7 CTD and show that this domain contains an xRRM, a class of atypical RRM first identified in the Tetrahymena thermophila telomerase LARP7 protein p65. The xRRM binds the 3′ end of 7SK RNA at the top of stem-loop 4 (SL4) and interacts with both unpaired and base-paired nucleotides. This study confirms that the xRRM is general to the LARP7 family of proteins and defines the binding site for hLARP7 on the 7SK RNA, providing insight into function.

Structure ofS. pombetelomerase protein Pof8 C-terminal domain is an xRRM conserved among LARP7 proteins

“…The structure of Pof8 RRM2 reported 12 here is the third example from the LARP7 family, and provides new insights into RNA binding 13 by these domains and those of the related La proteins. The xRRM was first structurally 14 characterized in p65 in the absence and presence of its RNA target (31), and subsequently in 15 human Larp7 (42,49). Comparing the three LARP7 RRM2 structures shows that they share all 16 the features of an xRRM first defined for p65 except for the length and sequence of helix a3that 17 extends beyond the b-sheet, which are strikingly variable (Fig 4).…”

“…The combination of helix a3, that lies across the b-sheet and over where the absent RNP2 6 and RNP1 would be, and conserved RNP3 sequence on the Pof8 RRM2 is unique to the xRRM 7 class of atypical RRMs, and led us to hypothesize that the Pof8 RRM2 is an xRRM. The human 8 genuine La protein also contains an atypical RRM2 that we previously predicted to be an xRRM 9 (49). Below we compare the conserved structural and RNA recognition features for the existing 10 examples of defined xRRMs (p65 and hLarp7) and predicted xRRMs (Pof8 and hLa).…”

“…In free hLarp7 a3 has 5 turns and is extended by an additional 20 turn on binding RNA. For both p65 and hLarp7 xRRMs, truncation of these extra turns of helix 21 a3 resulted in significantly reduced binding affinity to substrate RNA (31,49), indicating that the 22 region of helix a3 that extends beyond the b-sheet contributes to high affinity binding. 23…”

“…The Pof8 a3 is three turns long and ends at the protein C-terminus with two 1 consecutive lysines, the last of which is disordered. It seems likely that the short Pof8 helix a3 2 will result in a weaker binding affinity to the xRRMs of p65 (30 nM) (31) and hLarp7 (100 nM) 3 (42,49) to their cognate substrates. Additional affinity may be provided by interactions between 4 conserved residues on a1 and RNA.…”

Recombinant expression and purification of the RNA-binding LARP6 proteins from fish genetic model organisms

“…Of the five La-related protein subfamilies, the identification of the biochemical and cellular functions of LARP6 has been the most elusive. The Genuine La protein (now called LARP3) binds to the 3′ oligopyrimidine end of pre-tRNAs to assist maturation[2]; LARP7 similarly binds to the 3′ end of lncRNAs [3]. LARP1 and LARP4 bind to the 5′ and 3′ untranslated regions of mRNAs to regulate translation and degradation [4-7].…”